Use of immunesuppressant receptor

ABSTRACT

The present invention relates to use of an antagonist of BIR1 (B cell immunoglobulin receptor 1) related to the present invention, a method for screening the antagonist, in addition to subtype polypeptides of BIR1, the polynucleotide encoding them and antibodies for the polypeptides. BIR1 functions as an immunosuppressive receptor, and the antagonist of BIR1 has immunopotentive activity, which is able to use for preventing and/or treating a cancer, an immunodeficiency disease or an infectious disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 11/666,015filed Apr. 23, 2007, which is a National Stage Application filed under§371 of PCT Application No. PCT/JP2005/019272 filed Oct. 20, 2005. Theentire disclosures of the prior applications are considered part of thedisclosure of the continuation application and are hereby incorporatedby reference.

TECHNICAL FIELD

The present invention relates to use of an antagonist of theimmunosuppressive receptor related to the present invention.Specifically, the present invention relates to an immunopotentiatorcomprising the antagonist thereof and a method for screening theantagonist.

BACKGROUND OF THE INVENTION

It is known that molecules which function as immunosuppressive receptorsincludes CTLA-4 (N. Engl. J. Med., 338(25): 1813-21 (1998)), PD-1(Proc.Natl. Acad. Sci. USA, 98(24): 13866-71 (2001)), FcγRIIB (Science,290(5489): 84-9 (2000)) and the like.

An amino acid sequence of the protein related to the present invention(sometimes referred to as B cell immunoglobulin receptor 1 (BIR1)hereinafter) and a nucleotide sequence of DNA encoding it are reportedin WO 99/33873 pamphlet. However, not only functions of the proteinrelated to the present invention are not sufficiently elucidated, usesof antagonists and agonists thereof are not known at all.

[Non-patent Reference 1] N. Engl. J. Med., 338(25): 1813-21 (1998)

[Non-patent Reference 2] Proc. Natl. Acad. Sci. USA, 98(24): 13866-71(2001)

[Non-patent Reference 3] Science, 290(5489): 84-9 (2000)

[Patent Reference 1] WO 99/33873 pamphlet

DISCLOSURE OF THE INVENTION Problems That the Invention is To Solve

Problems of the present invention are to elucidate functions of thereceptor related to the present invention and to find uses ofantagonists or agonists.

Means For Solving the Problems

In order to solve the problems, the inventors of the present applicationhave conducted intensive studies and, as a result, elucidated for thefirst time that the receptor related to the present invention functionsas an immunosuppressive receptor, and have found respective uses ofantagonists and agonists. The inventors of the present application alsofound a method for screening the antagonists and agonists, therebyaccomplishing the present invention.

Namely, the present invention relates to

(1) an immunopotentiator which comprises an antagonist of a proteinhaving an amino acid sequence identical to or substantially identical toan amino acid sequence selected from the amino acid sequencesrepresented by SEQ ID NOs:1 to 6,

(2) the immunopotentiator according to the aforementioned (1), whereinthe antagonist is a substance which attenuates or inhibits suppressionof intracellular signal transduction by the protein described in theaforementioned (1),

(3) the immunopotentiator according to the aforementioned (2), whereinthe substance described in the aforementioned (2) is a substance whichinhibits a ligand bond of the protein described in the aforementioned(1),

(4) the immunopotentiator according to the aforementioned (2), whereinthe substance described in the aforementioned (2) is a substance whichinhibits binding of a phosphatase to an intracellular region of theprotein described in the aforementioned (1),

(5) the immunopotentiator according to the aforementioned (2), whereinthe substance described in the aforementioned (2) is a substance whichinhibits phosphatase activity,

(6) the agent according to the aforementioned (3), wherein the substancewhich inhibits a ligand bond is a protein or polypeptide comprising anoptional region in the extracellular region of the protein described inthe aforementioned (1) or an antibody therefor,

(7) the immunopotentiator according to the aforementioned (6), whereinthe aforementioned protein or polypeptide described in (6) is a proteinor polypeptide comprising an amino acid sequence identical to orsubstantially identical to an amino acid sequence selected from aminoacids 1 to 227 in the amino acid sequence represented by SEQ ID NO:1,amino acids 1 to 227 in the amino acid sequence represented by SEQ IDNO:2, amino acids 1 to 132 in the amino acid sequence represented by SEQID NO:3, amino acids 1 to 137 in the amino acid sequence represented bySEQ ID NO:4, amino acids 1 to 42 in the amino acid sequence representedby SEQ ID NO:5 and amino acids 1 to 132 in the amino acid sequencerepresented by SEQ ID NO:6,

(8) the immunopotentiator according to the aforementioned (4) or (5),wherein the phosphatase is SHP-1, SHP-2, SHIP-1 and/or SHIP-2,

(9) the immunopotentiator according to the aforementioned (1), which isan agent for preventing and/or treating a disease selected from acancer, an immunodeficiency disease and an infectious disease,

(10) A method for screening an antagonist of a protein having an aminoacid sequence identical to or substantially identical to an amino acidsequence selected from the amino acid sequences represented by SEQ IDNOs:1 to 6, which comprises:

(i) allowing a protein or polypeptide comprising its extracellularregion to contact with a ligand in the presence of a compound to betested;

(ii) detecting a signal which is changed by the binding of the proteinor polypeptide comprising its extracellular region with the ligand; and

(iii) screening a compound which inhibits the binding, by comparingsignal strengths (ii) in the presence and absence of the compound to betested,

(11) A method for screening an antagonist of a protein having an aminoacid sequence identical to or substantially identical to an amino acidsequence selected from the amino acid sequences represented by SEQ IDNOs:1 to 6, which comprises:

(i) allowing a protein or polypeptide comprising its intracellularregion to contact with a phosphatase in the presence of a compound to betested;

(ii) detecting a signal which is changed by the binding of the proteinor polypeptide comprising its intracellular region with the phosphatase;and

(iii) screening a compound which inhibits the binding, by comparingsignal strengths in (ii) in the presence and absence of the compound tobe tested,

(12) A method for screening an antagonist of a protein having an aminoacid sequence identical to or substantially identical to an amino acidsequence selected from the amino acid sequences represented by SEQ IDNOs:1 to 6, which comprises:

(i) allowing a cell capable of expressing a protein having an amino acidsequence identical to or substantially identical to an amino acidsequence selected from the amino acid sequences represented by SEQ IDNOs:1 to 6, or a protein or polypeptide comprising its extracellularregion, to contact with a cell capable of expressing a ligand, or theligand, in the presence of a compound to be tested;

(ii) detecting a signal which is changed by the contact in (ii); and

(iii) comparing signal strengths of the step (ii) in the presence andabsence of the compound to be tested,

(13) A method for immunopotentiation, which comprises administering to amammal an effective amount of an antagonist of a protein having an aminoacid sequence identical to or substantially identical to an amino acidsequence selected from the amino acid sequences represented by SEQ IDNOs:1 to 6,

(14) use of an antagonist of a protein having an amino acid sequenceidentical to or substantially identical to an amino acid sequenceselected from the amino acid sequences represented by SEQ ID NOs:1 to 6,for producing an immunopotentiator,

(15) a method for preventing and/or treating a disease selected from acancer, an immunodeficiency disease and an infectious disease, whichcomprises administering to a mammal an effective amount of an antagonistof a protein having an amino acid sequence identical to or substantiallyidentical to an amino acid sequence selected from the amino acidsequences represented by SEQ ID NOs:1 to 6,

(16) use of an antagonist of a protein having an amino acid sequenceidentical to or substantially identical to an amino acid sequenceselected from the amino acid sequences represented by SEQ ID NOs:1 to 6,for producing an agent for preventing and/or treating a disease selectedfrom a cancer, an immunodeficiency disease and an infectious disease,

(17) a protein having an amino acid sequence identical to orsubstantially identical to one amino acid sequence selected from theamino acid sequences represented by SEQ ID NOs:3 to 6, or a partialpeptide thereof,

(18) a polynucleotide which encodes a protein having an amino acidsequence identical to or substantially identical to one amino acidsequence selected from the amino acid sequences represented by SEQ IDNOs:3 to 6, or a partial peptide thereof,

(19) an antibody for a protein having an amino acid sequence identicalto or substantially identical to one amino acid sequence selected fromthe amino acid sequences represented by SEQ ID NOs:3 to 6, or a partialpeptide thereof,

(20) an immunosuppressant, which comprises an agonist of a proteinhaving an amino acid sequence identical to or substantially identical toan amino acid sequence selected from the amino acid sequencesrepresented by SEQ ID NOs:1 to 6,

(21) the agent according to the aforementioned (20), wherein the agonistis a substance which keeps or reinforces suppression of intracellularsignal transduction by the protein described in the aforementioned (20),

(22) the agent according to the aforementioned (21), wherein thesubstance described in the aforementioned (21) is a substance whichcross-links the protein described in the aforementioned (20) with areceptor expressed in a cell which expresses the protein,

(23) the agent according to the aforementioned (21), wherein thesubstance described in the aforementioned (21) is an agonist antibodyfor the protein described in the aforementioned (20),

(24) the agent according to the aforementioned (20), which is an agentfor preventing and/or treating a disease selected from an autoimmunedisease, a rejection reaction after organ transplantation, an allergicdisease and an inflammatory disease,

(25) the agent according to the aforementioned (24), wherein theautoimmune disease is a disease having high value of lupusanticoagulation factor, systemic lupus erythematodes, articularrheumatism, multiple sclerosis or Takayasu arteritis,

(26) a method for immunosuppression, which comprises administering to amammal an effective amount of an agonist of a protein having an aminoacid sequence identical to or substantially identical to an amino acidsequence selected from the amino acid sequences represented by SEQ IDNOs:1 to 6,

(27) use of an agonist of a protein having an amino acid sequenceidentical to or substantially identical to an amino acid sequenceselected from the amino acid sequences represented by SEQ ID NOs:1 to 6,for producing an immunosuppressant,

(28) a method for preventing and/or treating a disease selected from anautoimmune disease, a rejection reaction after organ transplantation, anallergic disease and an inflammatory disease, which comprisesadministering to a mammal an effective amount of an agonist of a proteinhaving an amino acid sequence identical to or substantially identical toan amino acid sequence selected from the amino acid sequencesrepresented by SEQ ID NOs:1 to 6, and

(29) use of an agonist of a protein having an amino acid sequenceidentical to or substantially identical to an amino acid sequenceselected from the amino acid sequences represented by SEQ ID NOs:1 to 6,for producing an agent for preventing and/or treating a disease selectedfrom an autoimmune disease, a rejection reaction after organtransplantation, an allergic disease and an inflammatory disease.

Effect of the Invention

The antagonist of the protein related to the present invention isexpected to have effect to prevent and/or treat a cancer, animmunodeficiency disease or an infectious disease. The agonist of theprotein related to the present invention is also expected to have effectto prevent and/or treat an autoimmune disease, a rejection reactionafter organ transplantation, an allergic disease or an inflammatorydisease.

BEST MODE FOR CARRYING OUT THE INVENTION

According to the description of the present invention, the “proteinhaving substantially identical amino acid sequence” in the “proteinhaving an amino acid sequence identical to or substantially identical toan amino acid sequence selected from the amino acid sequencesrepresented by SEQ ID NOs:1 to 6” means a protein having the samefunction of the protein having an amino acid sequence selected from theamino acid sequences represented by SEQ ID NOs:1 to 6, in which severalamino acids (preferably from 1 to 5 residues, more preferably 1 or 2residues) of the amino acids selected from the amino acid sequencesrepresented by SEQ ID NOs:1 to 6 are deleted, several amino acids(preferably from 1 to 5 residues, more preferably 1 or 2 residues)therein are substituted with other amino acids, or several residues ofamino acids (preferably from 1 to 5 residues, more preferably 1 or 2residues) are added to or inserted into the amino acid sequence, orwhich has an amino acid sequence of the combination therewith. In thiscase, the position of the aforementioned deletion, substitution oraddition or insertion of amino acids is not particularly limited.Hereinafter, the “protein having an amino acid sequence identical to orsubstantially identical to an amino acid sequence selected from theamino acid sequences represented by SEQ ID NOs:1 to 6” as used in thespecification of the present invention may be called “the proteinrelated to the present invention” in some cases.

In this connection, the protein related to the present invention ispreferably a protein having an amino acid sequence identical to orsubstantially identical to an amino acid sequence selected from theamino acid sequences represented by SEQ ID NOs:1 to 4 and SEQ ID NO:6,more preferably a protein having an amino acid sequence identical to orsubstantially identical to an amino acid sequence selected from theamino acid sequences represented by SEQ ID NOs:1 to 4, furtherpreferably a protein having an amino acid sequence identical to orsubstantially identical to an amino acid sequence selected from theamino acid sequences represented by SEQ ID NO:1 or 2, and still furtherpreferably a protein having the amino acid sequence represented by SEQID NO:1 or 2.

According to the specification of the present invention, the “antagonistof a protein having an amino acid sequence identical to or substantiallyidentical to an amino acid sequence selected from the amino acidsequences represented by SEQ ID NOs:1 to 6” represents a substance whichattenuates or inhibits suppression of intracellular signal transductionby the protein related to the present invention. Such a substanceincludes

(i) a substance which inhibits ligand binding to the protein related tothe present invention and does not have agonist activity for the proteinrelated to the present invention;

(ii) a substance which inhibits binding of a phosphatase to theintracellular region of the protein related to the present invention;

(iii) a substance which inhibits activity of the phosphatase; and

(iv) a substance which inhibits activity of a phosphatase bound to theintracellular region of the protein related to the present invention;and the like.

In this case, the “intracellular signal” includes, intracellular signalsgenerated from B cell receptor (BCR) or B cell receptor complexes (BCR,CD79A (EMBO J., 7(11): 3457-3464 (1988)) and CD79B (Eur. J. Immunol.,22(6): 1621-1625 (1992))), activated Fc receptors (e.g., FcγRI (J. Biol.Chem., 266(20): 13449-13455 (1991))), CD14/TLR4 complex (Nature, 406:780-785 (2000)), FcεRI (Proc. Natl. Acad. Sci. USA, 85: 1907-1911(1988)) and the like. Additionally, the “suppression of intracellularsignal transduction by the protein related to the present invention”includes, for example, dephosphorylation of an intracellular signaltransduction-carrying molecule by a phosphatase bound to theintracellular region of the protein related to the present invention.

In this case, the “substance which inhibits ligand bond to the proteinrelated to the present invention and does not have agonist activity forthe protein related to the present invention” includes, for example, aprotein, a polypeptide or peptide, an antibody, a non-peptide compound,an organic synthesis compound or natural product (e.g., a fermentationproduct, a cell extract, a plant extract, an animal tissue extract orthe like) and the like.

Preferred as such a substance is a protein, a polypeptide or anantibody, and its illustrative examples include a protein or polypeptidecomprising an optional region in the extracellular region of the proteinrelated to the present invention and an antibody for the same and thelike.

The “protein or polypeptide comprising an optional region in theextracellular region of the protein related to the present invention” isa protein or polypeptide which contains an optional region in theextracellular region of the protein related to the present invention,which does not contain transmembrane region and intracellular region.Specifically, it is a protein or polypeptide which contains a regionoptionally selected from the region of amino acids 1 to 227 in the aminoacid sequence represented by SEQ ID NO:1, a region optionally selectedfrom the region of amino acids 1 to 227 in the amino acid sequencerepresented by SEQ ID NO:2, a region optionally selected from the regionof amino acids 1 to 132 in the amino acid sequence represented by SEQ IDNO:3, a region optionally selected from the region of amino acids 1 to137 in the amino acid sequence represented by SEQ ID NO:4, a regionoptionally selected from the region of amino acids 1 to 42 in the aminoacid sequence represented by SEQ ID NO:5 or a region optionally selectedfrom the region of amino acids 1 to 132 in the amino acid sequencerepresented by SEQ ID NO:6, and which does not contain transmembraneregion and intracellular region. The “transmembrane region” is a regionof amino acids 228 to 250 in the amino acid sequence represented by SEQID NO:1, a region of amino acids 228 to 250 in the amino acid sequencerepresented by SEQ ID NO:2, a region of amino acids 133 to 155 in theamino acid sequence represented by SEQ ID NO:3, a region of amino acids138 to 160 in the amino acid sequence represented by SEQ ID NO:4, aregion of amino acids 43 to 65 in the amino acid sequence represented bySEQ ID NO:5 or a region of amino acids 133 to 155 in the amino acidsequence represented by SEQ ID NO:6, and the “intracellular region” is aregion of amino acids 251 to 343 of the amino acid sequence representedby SEQ ID NO:1, a region of amino acids 251 to 343 in the amino acidsequence represented by SEQ ID NO:2, a region of amino acids 156 to 248in the amino acid sequence represented by SEQ ID NO:3, a region of aminoacids 161 to 253 in the amino acid sequence represented by SEQ ID NO:4,a region of amino acids 66 to 158 in the amino acid sequence representedby SEQ ID NO:5 or a region of amino acids 156 to 176 in the amino acidsequence represented by SEQ ID NO:6.

Those which are fused with other protein or polypeptide are alsoincluded in the protein or polypeptide containing an optional region inthe extracellular region of the protein related to the presentinvention. Examples include those which are fused with the Fc domain ofimmunoglobulin for the solubilization of the protein or polypeptide andthe like, and they can be produced by conventionally known methods.

The “optional region” in the “protein or polypeptide containing anoptional region in the extracellular region of the protein related tothe present invention” may be any region in the extracellular region ofthe protein related to the present invention, so long as it has theantagonist activity for the protein related to the present invention.

These proteins or polypeptides also include, for example, those in whichseveral amino acids (preferably from 1 to 5 residues, more preferably 1or 2 residues) in the protein or polypeptide are deleted, several aminoacids (preferably from 1 to 5 residues, more preferably 1 or 2 residues)therein are substituted with other amino acids, or several amino acids(preferably from 1 to 5 residues, more preferably 1 or 2 residues) areadded to or inserted into the amino acid sequence, or which have anamino acid sequence of the combination thereof, for the purpose ofkeeping or improving the antagonist activity, stabilizing the protein orpolypeptide or reducing antigenicity.

The protein or polypeptide containing an optional region in theextracellular region of the protein related to the present invention canbe prepared by conventionally known protein expression methods andpurification methods or by the methods described in Examples.

The “ligand” in the “ligand bond of the protein related to the presentinvention” is an intravital substance which binds to the extracellularregion of the protein related to the present invention and has theactivity to induce functions of the protein related to the presentinvention. A protein is preferred as such a ligand. The “extracellularregion of the protein related to the present invention” is a regionwhich comprises a domain so-called immunoglobulin or immunoglobulin-likedomain, and is a region of amino acids 1 to 227 in the amino acidsequence represented by SEQ ID NO:1, a region of amino acids 1 to 227 inthe amino acid sequence represented by SEQ ID NO:2, a region of aminoacids 1 to 132 in the amino acid sequence represented by SEQ ID NO:3, aregion of amino acids 1 to 137 in the amino acid sequence represented bySEQ ID NO:4, a region of amino acids 1 to 42 in the amino acid sequencerepresented by SEQ ID NO:5 or a region of amino acids 1 to 132 in theamino acid sequence represented by SEQ ID NO:6.

The term “does not have agonist activity for the protein related to thepresent invention” means that it does not have any activity whichstimulates functions of the protein related to the present invention.

The “intracellular region of the protein related to the presentinvention” is specifically a region of amino acids 251 to 343 in theamino acid sequence represented by SEQ ID NO:1, a region of amino acids251 to 343 in the amino acid sequence represented by SEQ ID NO:2, aregion of amino acids 156 to 248 in the amino acid sequence representedby SEQ ID NO:3, a region of amino acids 161 to 253 in the amino acidsequence represented by SEQ ID NO:4, a region of amino acids 66 to 158in the amino acid sequence represented by SEQ ID NO:5 or a region ofamino acids 156 to 176 in the amino acid sequence represented by SEQ IDNO:6. So-called ITIM (immunoreceptor tyrosine-based inhibitorymotif)-like domain is present in the intracellular region of the proteinrelated to the present invention, which, specifically, is present inamino acids 311 to 316 and/or 336 to 341 in the amino acid sequencerepresented by SEQ ID NO:1, amino acids 311 to 316 and/or 336 to 341 inthe amino acid sequence represented by SEQ ID NO:2, amino acids 216 to221 and/or 241 to 246 in the amino acid sequence represented by SEQ IDNO:3, amino acids 221 to 226 and/or 246 to 251 in the amino acidsequence represented by SEQ ID NO:4, amino acids 126 to 131 and/or 151to 156 in the amino acid sequence represented by SEQ ID NO:5 or aminoacids 169 to 174 in the amino acid sequence represented by SEQ ID NO:6.

The “phosphatase” in the “substance which inhibits binding of aphosphatase to the intracellular region of the protein related to thepresent invention”, the “substance which inhibits the phosphataseactivity” or the “substance which inhibits activity of a phosphatasebound to the intracellular region of the protein related to the presentinvention”, includes, for example, phosphatases called SHP-1 (Nature,352(6337): 736-739 (1991)), SHP-2 (Proc. Natl. Acad. Sci., 90: 2197-2201(1993)), SHIP-1 (Proc. Natl. Acad. Sci., 93: 1689-1693 (1996)), SHIP-2(Biochem. Biophys. Res. Commun., 239(3): 697-700 (1997)) and the like.

The “binding of a phosphatase to the intracellular region” in the“substance which inhibits binding of a phosphatase to the intracellularregion of the protein related to the present invention” is the bindingof a phosphatase to the ITIM-like domain, and the binding requiresphosphorylation of certain tyrosine residues contained in the ITIM-likedomain. Such tyrosine residues is amino acid 313th position and 338thposition tyrosine residues in the amino acid sequence represented by SEQID NO:1, amino acid 313th position and 338th position tyrosine residuesin the amino acid sequence represented by SEQ ID NO:2, amino acid 218thposition and 243th position tyrosine residues in the amino acid sequencerepresented by SEQ ID NO:3, amino acid 223th position and 248th positiontyrosine residues in the amino acid sequence represented by SEQ ID NO:4,amino acid 128th position and 153th position tyrosine residues in theamino acid sequence represented by SEQ ID NO:5 or amino acid 171thposition tyrosine residue in the amino acid sequence represented by SEQID NO:6.

The target to be dephosphorylated by the phosphatase in the “substancewhich inhibits the phosphatase activity” or the “substance whichinhibits activity of a phosphatase bound to the intracellular region ofthe protein related to the present invention” includes, for example,intracellular signal transducer molecules such as a kinase, an inositolphosphate and the like. The kinase includes, for example, aserine/threonine kinase (e.g., protein kinase A, protein kinase C,Ca²⁺/calmodulin dependent protein kinase, MAP kinase, Mos/Raf kinase orthe like), a tyrosine kinase (e.g., receptor type tyrosine kinase,non-receptor type tyrosine kinase or the like) and the like. Theinositol phosphate includes, for example,phosphatidylinositol-3,4,5-triphosphate and the like.

The target kinase of the phosphatase includes, for example, ERK 2(Biochem. Biophys. Res. Commun., 182: 14161422 (1992)), BTK (Nature, 361(6409), 226-233 (1993)) and SYK (J. Biol. Chem., 266: 15790-15796(1991)), JAK 1 (Molec. Cell. Biol., 2057-2065 (1991)), LAK 2 (Biochem.Biophys. Res. Commun., 246: 627-633 (1998)), JAR 3 (Proc. Natl. Acad.Sci., 91: 6374-6378 (1994)), ZAP 70 (Cell, 71: 649-662 (1992)), BLNK(Immunity, 9: 93-103 (1998)), FYN (Proc. Natl. Acad. Sci. USA, 83(15):5459-5463 (1986)), LCK (Biochim. Biophys. Acta, 888(3): 286-295 (1986))and the like, of which ERK 2 is more preferable.

The “antibody for a protein or polypeptide comprising an optional regionin the extracellular region of the protein related to the presentinvention” may be any antibody such as a human antibody, a mouseantibody, a rat antibody, a domestic fowl antibody, a rabbit antibody ora goat antibody, as long as it inhibits binding of a ligand to theprotein related to the present invention and does not have agonistactivity for the protein related to the present invention. It may alsobe any one of their polyclonal or monoclonal antibodies, complete typeor shortened type (e.g., F(ab′)₂, Fab′, Fab and Fv fragment and thelike) antibodies, chimeric antibodies, humanized antibodies or completehuman antibodies. Such antibodies can be produced by conventionallyknown antibody or antiserum production methods or in accordance with themethods described in Examples, wherein a protein or polypeptidecomprising an optional region in the extracellular region of the proteinrelated to the present invention is used as the antigen. The protein orpolypeptide comprising an optional region in the extracellular region ofthe protein related to the present invention can be produced byconventionally known protein expression and purification methods or inaccordance with the methods described in Examples.

The aforementioned substance which attenuates or inhibits suppression ofintracellular signal transduction by the protein related to the presentinvention is useful as an agent for preventing and/or treating a cancer,an immunodeficiency disease or an infectious disease.

According to the specification of the present invention, the “agonist ofa protein having an amino acid sequence identical to or substantiallyidentical to an amino acid sequence selected from the amino acidsequences represented by SEQ ID NOs:1 to 6” means a substance whichkeeps or reinforces suppression of intracellular signal transduction bythe protein related to the present invention. Such substance includes

(i) a substance which cross-links the protein related to the presentinvention with a receptor expressed in a cell which expresses theprotein;

(ii) an agonist antibody for the protein related to the presentinvention; and

(iii) a ligand for the protein related to the present invention and thelike.

In this connection, the “intracellular signal” includes, for example,intracellular signals generated from a B cell receptor or a B cellreceptor complex, an activated Fc receptor, a CD14/TLR4 complex, anFcεRI and the like. Additionally, the “to keep or reinforce suppressionof intracellular signal transduction by the protein related to thepresent invention” includes, for example, maintenance or increase offrequency of the dephosphorylated state of an intracellular signaltransduction-carrying molecule by a phosphatase bound to theintracellular region of the protein related to the present invention andthe like.

In this connection, the “receptor” in the “substance which cross-linksthe protein related to the present invention with a receptor expressedin a cell which expresses the protein” includes, for example, a B cellreceptor or a B cell receptor complex, an activated Fc receptor, aCD14/TLR4 complex, an FcεRI and the like.

The “substance which cross-links the protein related to the presentinvention with a receptor expressed in a cell which expresses theprotein” as the substance which keeps or reinforces suppression ofintracellular signal transduction by the protein related to the presentinvention includes, for example, a protein, a polypeptide, an antibodyand the like. The substance is preferably an antibody which recognizesboth of the protein related to the present invention and a receptorexpressed in a cell which expresses the protein.

The “agonist antibody for the protein related to the present invention”as the substance which keeps or reinforces suppression of intracellularsignal transduction by the protein related to the present invention maybe any antibody such as a human antibody, a mouse antibody, a ratantibody, a domestic fowl antibody, a rabbit antibody or a goatantibody, so long as it is an antibody which activates the proteinrelated to the present invention, and it may also be any one ofpolyclonal or monoclonal antibodies, complete type or shortened type(e.g., F(ab′)₂, Fab′, Fab and Fv fragment and the like) antibodies,chimeric antibodies, humanized antibodies or complete human antibodies.Such antibodies can be produced by conventionally known antibody orantiserum production methods, or in accordance with the methodsdescribed in Examples wherein a protein or polypeptide comprising anoptional region in the extracellular region of the protein related tothe present invention is used as the antigen. The protein or polypeptidecomprising an optional region in the extracellular region of the proteinrelated to the present invention can be produced by conventionally knownprotein expression and purification methods.

The “ligand for the protein related to the present invention” as thesubstance which keeps or reinforces suppression of intracellular signaltransduction by the protein related to the present invention is anintravital substance which binds to the extracellular region of theprotein related to the present invention and has the activity to inducefunctions of the protein related to the present invention, and apolypeptide having its partial peptide is also included therein whensuch a substance is a protein.

As described the above, the substance which keeps or reinforcessuppression of intracellular signal transduction by the protein relatedto the present invention is useful as an agent for preventing and/ortreating an autoimmune disease, a rejection reaction after organtransplantation, an allergic disease or an inflammatory disease.

Screening Method of the Invention

According to the specification of the present invention, the method forscreening an antagonist or agonist of the protein related to the presentinvention (sometimes referred to as screening method of the presentinvention hereinafter) can be carried out, for example, using afluorescence polarization homogenous assay, a time-resolved fluorescenceassay, a fluorescence resonance energy transfer assay, a chemicalamplification type luminescence proximity homogenous assay, an RI assay,a bioluminescence resonance energy transfer assay, a two hybrid reportergene assay, an intracellular Ca²⁺ concentration measuring assay, anELISA assay or the like.

The “protein or polypeptide comprising the extracellular region of theprotein related to the present invention” in the method for screening anantagonist of the protein related to the present invention is apolypeptide comprising a region optionally selected from the region ofamino acids 1 to 227 in the amino acid sequence represented by SEQ IDNO:1, a region optionally selected from the region of amino acids 1 to227 in the amino acid sequence represented by SEQ ID NO:2, a regionoptionally selected from the region of amino acids 1 to 132 in the aminoacid sequence represented by SEQ ID NO:3, a region optionally selectedfrom the region of amino acids 1 to 137 in the amino acid sequencerepresented by SEQ ID NO:4, a region optionally selected from the regionof amino acids 1 to 42 in the amino acid sequence represented by SEQ IDNO:5 or a region optionally selected from the region of amino acids 1 to132 in the amino acid sequence represented by SEQ ID NO:6. In thisconnection, the “region being optionally selected” means a region whichis necessary for keeping the activity of the protein related to thepresent invention to bind to a ligand.

The “protein or polypeptide comprising the intracellular region of theprotein related to the present invention” is a protein or polypeptidewhich comprises the region of the aforementioned ITIM-like domain. Inthis connection, the tyrosine residue contained in the ITIM-like domainmay be phosphorylated.

These polypeptides, those to which detection label such as an enzyme, afluorescent material, a fluorescent protein, a luminescent material or aradioisotope or the like is added can be used. These detection labelsmay be added after or before the binding of a protein or polypeptidecomprising the extracellular region of the protein related to thepresent invention to a ligand, via a substance or antibody capable ofrecognizing them. Specifically, the addition can be carried out bylabeling a protein or polypeptide comprising the extracellular region ofthe protein related to the present invention or a ligand with biotin,and adding the aforementioned detection label labeled with avidin to theother one. This is the same also in the case of the binding of a proteinor polypeptide comprising the intracellular region of the proteinrelated to the present invention with a phosphatase.

The enzyme as the detection label, includes, for example,β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malatedehydrogenase and the like. The fluorescent material as the detectionlabel, includes, for example, FITC (fluorescein isothiocyanate), PI(propidium iodide), Cy-Chrome, APC (allophycocyanine), R-PE(R-phycoerythrin), a fluorescent lanthanide chelate (e.g., europium,samarium, terbium, dysprosium or the like) and the like. The fluorescentprotein as the detection label includes, for example, GFP (greenfluorescent protein), AmCyan, ZsGreen, ZsYellow, AsRed, RCFP (reef coralfluorescent protein), DsRed, AcGFP1, HcRed1, CopGFP, PhiYFP-m. EYFP,KFP-Red and the like. The radioisotope as the detection label includes,for example, [³²P], [³H], [¹²⁵I], [³⁵S] and [¹⁴C]. The luminescentmaterial as the detection label includes, for example, luminol, luminolderivatives, luciferin, lucigenin and the like.

In the description of the present invention, the “ligand” in the “stepof allowing a protein or polypeptide comprising its extracellular regionto contact with a ligand” of the screening method of the presentinvention is an intravital substance which binds to the extracellularregion of the protein related to the present invention and has theactivity to induce functions of the protein related to the presentinvention. A protein is preferred as such a ligand.

Examples of the “signal which is changed by the binding of the proteinor polypeptide comprising its extracellular region of the proteinrelated to the present invention with a ligand” or the “signal which ischanged by the binding of the protein or polypeptide comprising itsintracellular region of the protein related to the present inventionwith a phosphatase” includes a fluorescence generated by theaforementioned fluorescent material or fluorescent protein, a radiationfrom the radioisotope and the like as the detection labels.Additionally, development of a color by the reaction of theaforementioned enzyme as the detection label with a color developingsubstrate can also be exemplified. As the aforementioned enzyme,fluorescent material, fluorescent protein, luminescent material,radioisotope and color developing substrate, commercially availableproducts can be used.

Although the “cell which expresses a protein having an amino acidsequence identical to or substantially identical to an amino acidsequence selected from the amino acid sequences represented by SEQ IDNOs:1 to 6” may be any cell which expresses the protein related to thepresent invention, it is preferably a cell transiently or steadilytransformed by an expression vector capable of expressing the proteinrelated to the present invention. As such an expression vector, aproduct which is generally put on the market can be used. Additionally,the cell to be transformed includes, for example, simian COS-1 cell,COS-7 cell, Chinese hamster CHO cell, human HEK 293T cell, U937 cell,Jurkat cell, HELA cell, Daudi cell, K562 cell, mouse L cell and thelike.

Although the “cell which expresses a ligand” may be any cell whichexpresses the ligand, it is preferably a cell transiently or steadilytransformed by an expression vector capable of expressing the ligand. Assuch an expression vector, a product which is generally put on themarket can be used, and the aforementioned cell can be used as the cellto be transformed.

The “signal which is changed when a cell which expresses a proteinhaving an amino acid sequence identical to or substantially identical toan amino acid sequence selected from the amino acid sequencesrepresented by SEQ ID NOs:1 to 6 is allowed to contact with a cell thatexpresses a ligand or the ligand” includes, for example, signals (e.g.,color development, fluorescence, luminescence, radiation and the like)corresponding to the intracellular cAMP concentration of such cell,intracellular Ca²⁺ concentration, IP₃ concentration, phosphorylation ordephosphorylation of an intracellular signal transducer molecule (e.g.,Erk 2 or the like), binding of a phosphatase to the protein related tothe present invention, amount of a produced cytokine (e.g., interleukin2 (IL-2) or the like) and the like.

As a screening method of the present invention, it can be carried outillustratively by the following method. Namely, an antibody for anactivated receptor (e.g., BCR) which is co-expressed with the proteinrelated to the present invention, an antibody for the protein related tothe present invention or a ligand of the protein related to the presentinvention is simultaneously immobilized on a carrier such as agarosebeads or a culture plate. When a cell expressing the protein related tothe present invention (BRI 1-overexpressing cell, a B cell strainco-expressing BCR and BIR1, B cell, monocyte, peripheral mononuclearcell or the like) is added to the aforementioned immobilized carrier orplate to carry out the stimulation, a compound to be tested (a lowmolecular compound, a peptide, a solubilized protein, an antibody or thelike) is added at the same time. After the culturing for a predeterminedperiod of time, an antagonist of the protein related to the presentinvention can be screened, wherein the amount of interleukin 2 (IL-2)secreted into the culture supernatant, intracellular Ca²⁺ concentration,phosphorylation of intracellular tyrosine residue of the protein relatedto the present invention, binding of a phosphatase to the proteinrelated to the present invention and phosphorylation of an Erk 2 or thelike intracellular signal transducer molecule are used as the index.

Additionally, a phosphorylated peptide comprising a region of theITIM-like domain of the protein related to the present invention isbiotinylated and allowed to bind to a carrier such as an agarose beadsor a culture plate to which an anti-biotin antibody was immobilized, anda compound to be tested (e.g., a low molecular compound, a peptide, asolubilized protein, an antibody or the like) and a lysate of A20IIA1.6cell are added thereto, followed by incubation for a predeterminedperiod of time. After the reaction with a primary antibody for SHP-1,SHP-2, SHIP-1 or SHIP-2, amount of the phosphatase bound to thephosphorylated peptide is measured using a secondary antibody. Acompound which reduces amount of the bound phosphatase can be screenedas an antagonist of the protein related to the present invention.

A method for screening a compound which decreases or increases theexpression level of the protein related to the present invention can becarried out, for example, by the following method. That is, cell (e.g.,BIR1-overexpressing cells, human monocyte cell strain THP-1 cells, U 937cells, CD 14-positive cells (monocytes) isolated from human peripheralblood, CD 19-positive cells (B cells) or the like) are inoculated onto aculture plate and cultured for a predetermined period of time togetherwith a compound to be tested (e.g., a low molecular compound, a peptide,a solubilized protein, an antibody or the like), in the presence orabsence of an inflammation stimulator (e.g., lipopolysaccharide (LPS),phorbol myristate acetate (PMA), interferon gamma (IFN-γ), TNF-α or thelike). RNA is extracted from the cells, and the expression level of theRNA is measured by quantitative RT-PCR using primers specific to theprotein related to the present invention. Alternatively, an anti-humanBIR1 monoclonal antibody is added to the cultured cells, then adding anFITC-labeled secondary antibody is added thereto to determine the BIR1expressed on the cell surface by a flow cytometer. Furthermore, a celllysate is prepared after the culturing, and the expression level of theprotein is measured by Western blotting using an antibody for theprotein related to the present invention.

Additionally, this can also be carried out by the following method.Specifically, an expression regulating region (e.g., promoter, enhancer,CAAT box, TATA box or the like), a DNA comprising 5′-non-translationregion and a region around translation initiation region and a reportergene (e.g., luciferase gene, chloramphenicol acetyltransferase (CAT)gene, β-galactosidase gene or the like) are connected with one another,and the thus prepared recombinant vector is transferred into appropriatecells. The cells are cultured in the presence or absence of a compoundto be tested, under such an environment that transcription of the BIR1gene can be effected to confirm transcription accelerating activity ortranscription regulating activity of the compound to be tested bymeasuring the expression level of said reporter gene. The expressionregulating region of BIR1 gene, 5′-non-translation region and a regionaround translation initiation region can be obtained by conventionallyknown methods.

Protein of the Invention

According to the specification of the present invention, the “proteinhaving a substantially identical amino acid sequence” in the “proteinhaving an amino acid sequence identical to or substantially identical toone amino acid sequence selected from the amino acid sequencesrepresented by SEQ ID NOs:3 to 6, or a partial peptide thereof” alsoincludes a protein having the same function of the protein having oneamino acid sequence selected from the amino acid sequences representedby SEQ ID NOs:3 to 6, wherein several amino acids (preferably from 1 to5 residues, more preferably 1 or 2 residues) in an amino acid sequenceselected from SEQ ID NOs:3 to 6 are deleted, several amino acids(preferably from 1 to 5 residues, more preferably 1 or 2 residues) inthe same are substituted with other amino acids, or several amino acids(preferably from 1 to 5 residues, more preferably 1 or 2 residues) areadded to or inserted into the amino acid sequence, or which has an aminoacid sequence of the combination thereof. In this case, the position ofthe aforementioned deletion, substitution or addition or insertion ofamino acids is not particularly limited. Hereinafter, the “proteinhaving an amino acid sequence identical to or substantially identical toone amino acid sequence selected from the amino acid sequencesrepresented by SEQ ID NOs:3 to 6” may be called in some cases “theprotein of the present invention”.

Additionally, the protein of the present invention includes an aminoacid sequence having a homology of about 70% or more, preferably about80% or more, more preferably about 90% or more, particularly preferablyabout 95% or more, and most preferably about 98% or more with the aminoacid sequence selected from SEQ ID NOs:3 to 6, over at least 20 residuesor more, preferably 50 residues or more, more preferably 100 residues ormore, and further preferably the entire region.

According to the protein in the specification of the present invention,the left terminal is the N-terminal (amino terminal) and the rightterminal is the C-terminal (carboxyl terminal) in accordance with theconventional peptide marking.

The C-terminal of the protein of the, present invention may be any oneof a carboxyl group, carboxylate, amido and ester (—COOR). In this case,as R in the ester, for example, a C1-6 alkyl group (e.g., methyl, ethyl,n-propyl, isopropyl, n-butyl or the like), a C3-8 cycloalkyl group(e.g., cyclopentyl, cyclohexyl or the like), a C6-12 aryl group (e.g.,phenyl, α-naphthyl or the like), a phenyl-C1-2 alkyl group (e.g.,benzyl, phenetyl or the like), an α-naphthyl-C1-2 alkyl group (e.g.,α-naphthylmethyl or the like), a C7-14 aralkyl group, pivaloyloxymethylgroup and the like are used.

When the protein of the present invention has a carboxyl group at otherthan the C-terminal, those in which the carboxyl group is amidated oresterificated are also included in the protein of the present invention.Ester in the case includes the ester in the C-terminal and the like asmentioned above.

Additionally, the protein of the present invention also includes theprotein those in which the amino group of an N-terminus amino acidresidue (e.g., methionine residue or the like) is protected with aprotecting group (e.g., a C1-6 acyl group (e.g., formyl group, acetylgroup or the like) or the like), those in which an N-terminus glutamineresidue formed by digestion in the living body is converted intopyroglutamic acid, those in which a substituent group (e.g., —OH, —SH,amino group, imidazole group, indole group, guanidino group or the like)on the side chain of an amino acid in the molecule is protected with anappropriate protecting group (e.g., a C1-6 acyl group (e.g., formylgroup, acetyl group or the like) or the like), a conjugated protein suchas a sugar chain-attached glycoprotein and the like.

The protein of the present invention is preferably a protein comprisingthe amino acid sequence represented by SEQ ID NO:3, a protein comprisingthe amino acid sequence represented by SEQ ID NO:4, a protein comprisingthe amino acid sequence represented by SEQ ID NO:5 or a proteincomprising the amino acid sequence represented by SEQ ID NO:6, and morepreferably a protein consisting of the amino acid sequence representedby SEQ ID NO:3, a protein consisting of the amino acid sequencerepresented by SEQ ID NO:4, a protein consisting of the amino acidsequence represented by SEQ ID NO:5 or a protein consisting of the aminoacid sequence represented by SEQ ID NO:6.

According to the specification of the present invention, the “partialpeptide” (sometimes referred to as partial peptide of the presentinvention hereinafter) in the “protein having an amino acid sequenceidentical to or substantially identical to an amino acid sequenceselected from the amino acid sequences represented by SEQ ID NOs:3 to 6,or a partial peptide thereof” includes, for example, an optional regionin the extracellular region of the protein of the present invention(e.g., a polypeptide comprising a region optionally selected from theregion of amino acids 1 to 132 in the amino acid sequence represented bySEQ ID NO:3, a region optionally selected from the region of amino acids1 to 137 in the amino acid sequence represented by SEQ ID NO:4, a regionoptionally selected from the region of amino acids 1 to 42 in the aminoacid sequence represented by SEQ ID NO:5 or a region optionally selectedfrom the region of amino acids 1 to 132 in the amino acid sequencerepresented by SEQ ID NO:6), and an optional region in the intracellularregion of the protein of the present invention (e.g., a regionoptionally selected from the region of amino acids 156 to 248 in theamino acid sequence represented by SEQ ID NO:3, a region optionallyselected from the region of amino acids 161 to 253 in the amino acidsequence represented by SEQ ID NO:4, a region optionally selected fromthe region of amino acids 66 to 158 in the amino acid sequencerepresented by SEQ ID NO:4 and a region optionally selected from theregion of amino acids 156 to 176 in the amino acid sequence representedby SEQ ID NO:6) and the like. In this connection, the “region optionallyselected” includes, for example, a region having an amino acid sequenceof at least 10 or more, preferably 20 or more, more preferably 50 ormore, most preferably 100 or more residues of the amino acid sequence ofthe present invention and the like.

Additionally, the partial peptide of the present invention also includesthose in which several amino acids (preferably from 1 to 5 residues,more preferably 1 or 2 residues) in the amino acid sequence are deleted,those in which several amino acids (preferably from 1 to 5 residues,more preferably 1 or 2 residues) therein are substituted with otheramino acids, or several amino acids (preferably from 1 to 5 residues,more preferably 1 or 2 residues) are added to or inserted into the aminoacid sequence, or those which have an amino acid sequence in combinationtherewith. When the amino acid is deleted, substituted, added orinserted in the amino acid sequence like the aforementioned case, theposition is not particularly limited.

The C-terminal of the partial peptide of the present invention may beany one of a carboxyl group, carboxylate, amido and ester (—COOR). Inthis connection, R of the ester includes similar groups described in theforegoing on the protein of the present invention. When the partialpeptide of the present invention has a carboxyl group at other than theC-terminus, the partial peptide of the present invention also includesthose in which the carboxyl group is amidated or esterificated. Theester in that case includes, for example, the aforementioned C-terminalester and the like.

Similarly to the case of the aforementioned protein of the presentinvention, the partial peptide of the present invention also includesthe peptide such as those in which the amino group of the N-terminusmethionine residue is protected with a protecting group, those in whichthe Gln formed by digestion of the N-terminal side in the living body isconverted into pyroglutamic acid, those in which a substituent group onthe side chain of an amino acid in the molecule is protected with anappropriate protecting group, or a complex protein such as a sugarchain-linked glycoprotein.

The salt of the protein of the present invention or a partial peptidethereof includes, a physiologically acceptable salt with an acid orbase. The acid addition salt includes, for example, a salt with aninorganic acid (e.g., hydrochloric acid, phosphoric acid, hydrobromicacid, sulfuric acid or the like) and a salt with an organic acid (e.g.,acetic acid, formic acid, propionic acid, fumaric acid, maleic acid,succinic acid, tartaric acid, citric acid, malic acid, oxalic acid,benzoic acid, methanesulfonic acid, benzenesulfonic acid or the like)and the like. The base addition salt includes, for example, a salt withammonium hydroxide, an alkali or alkaline earth metal hydroxide (e.g.,lithium hydroxide, sodium hydroxide, potassium hydroxide, calciumhydroxide, magnesium hydroxide or manganese hydroxide). Thephysiologically acceptable acid salt is especially preferred.

The partial peptide of the protein of the present invention is useful asan agent for preventing and/or treating, or diagnosing and/or testing acancer, an immunodeficiency disease or an infectious disease, as asubstance which inhibits binding of a ligand to the protein related tothe present invention. Also, it can be used in the screening of anantagonist or agonist of the protein related to the present invention.Additionally, by using the protein of the present invention or a partialpeptide thereof as the antigen, it can be also used for the preparationof an antibody for the protein of the present invention or a partialpeptide thereof.

Polynucleotide of the Invention

According to the description of the present invention, the“polynucleotide encoding a protein having an amino acid sequenceidentical to or substantially identical to one amino acid sequenceselected from the amino acid sequences represented by SEQ ID NOs:3 to 6,or a partial peptide thereof” may be any polynucleotide so long as ithas a nucleotide sequence encoding the protein of the present inventionor a partial peptide thereof. It is known that 1 to 6 codons encode oneamino acid, for example, TTT or TTC corresponds to Phe; TTA, TTG, CTT,CTC, CTA or CTG corresponds to Leu; ATT, ATC or ATA corresponds to Ile;ATG corresponds to Met; GTT, GTC, GTA or GTG corresponds to Val; TCT,TCC, TCA or TCG corresponds to Ser; CCT, CCC, CCA or CCG corresponds toPro; ACT, ACC, ACA or ACG corresponds to Thr; GCT, GCC, GCA or GCGcorresponds to Ala; TAT or TAC corresponds to Tyr; CAT or CACcorresponds to His; CAA or CAG corresponds to Gln; AAT or AACcorresponds to Asn; AAA or AAG corresponds to Lys; GAT or GACcorresponds to Asp corresponds GAA or GAG corresponds to Glu; TGT or TGCcorresponds to Cys; TGG corresponds to Trp; CGT, CGC, CGA or CGGcorresponds to Arg; AGT or AGC corresponds to Ser; AGA or AGGcorresponds to Arg; and GGT, GGC, GGA or GGG corresponds to Gly.Therefore, the polynucleotide coding for the protein of the presentinvention or a partial peptide thereof includes polynucleotides in whichrespective codons corresponding to respective amino acids are optionallycombined. Hereinafter, the polynucleotide encoding a protein having anamino acid sequence identical to or substantially identical to one aminoacid sequence selected from the amino acid sequences represented by SEQID NOs:3 to 6, or a partial peptide thereof, is sometimes referred to as“polynucleotide of the present invention”.

The polynucleotide of the present invention may be any one of a genomicDNA, a cDNA, a synthetic DNA, an RNA and a DNA-RNA hybrid.

According to the specification of the present invention, in addition tothe DNA having one nucleotide sequence selected from the nucleotidesequences represented by SEQ ID NOs:9 to 12, the polynucleotide of thepresent invention includes a polynucleotide having a nucleotide sequencewhich hybridizes with a complementary chain DNA of the DNA under astringent condition and encoding a protein having substantially the sameproperties of the protein of the present invention. The hybridizationcan be carried out in accordance with the conventionally known methods(Molecular Cloning (Sambrook, J., Fritsch, E. F., Maniatis, T., ColdSpring Harbor Laboratory Press) (1989), Gene, 10: 63 (1980) and thelike). The hybridization conditions can be determined by selectingtemperature, ionic strength, primer length and the like appropriately.In general, the stringency is increased when the temperature is high andthe ionic strength is law. The highly stringent conditions include, forexample, hybridization at 65° C. in a buffer containing 0.5 M NaHPO₄, 7%SDS and 1 mM EDTA and washing treatment at 65° C. in a buffer containing0.1×SSC and 0.1% SDS.

Such a polynucleotide includes, for example, a DNA comprising anucleotide sequence having a homology of 90% or more, preferably 95% ormore, more preferably 98% or more, with a DNA comprising one nucleotidesequence selected from the nucleotide sequences represented by SEQ IDNOs:9 to 12, over at least 20 bases, preferably 50 bases, morepreferably 100 bases, further preferably the entire region, and thelike.

Among the polynucleotides of the present invention, a polynucleotideencoding a protein having an amino acid sequence identical to orsubstantially identical to the amino acid sequences represented by SEQID NO:3, or a partial peptide thereof, is preferably a DNA comprisingthe nucleotide sequence represented by SEQ ID NO:9 or comprising itspartial sequence, more preferably a DNA comprising the nucleotidesequence represented by SEQ ID NO:9, further preferably a DNA consistingof the nucleotide sequence represented by SEQ ID NO:9.

Among the polynucleotides of the present invention, a polynucleotideencoding a protein having an amino acid sequence identical to orsubstantially identical to the amino acid sequences represented by SEQID NO:4, or a partial peptide thereof, is preferably a DNA comprisingthe nucleotide sequence represented by SEQ ID NO:10 or comprising itspartial sequence, more preferably a DNA comprising the nucleotidesequence represented by SEQ ID NO:10, further preferably a DNAconsisting of the nucleotide sequence represented by SEQ ID NO:10.

Among the polynucleotides of the present invention, preferred apolynucleotide encoding a protein having an amino acid sequenceidentical to or substantially identical to the amino acid sequencesrepresented by SEQ ID NO:5, or a partial peptide thereof, is preferablya DNA comprising the nucleotide sequence represented by SEQ ID NO:11 orcomprising its partial sequence, more preferably a DNA comprising thenucleotide sequence represented by SEQ ID NO:11, further preferably aDNA consisting of the nucleotide sequence represented by SEQ ID NO:11.

Among the polynucleotides of the present invention, a polynucleotideencoding a protein having an amino acid sequence identical to orsubstantially identical to the amino acid sequences represented by SEQID NO:6, or a partial peptide thereof, is preferably a DNA comprisingthe nucleotide sequence represented by SEQ ID NO:12 or comprising itspartial sequence, more preferably a DNA comprising the nucleotidesequence represented by SEQ ID NO:12, further preferably a DNAconsisting of the nucleotide sequence represented by SEQ ID NO:12.

The polynucleotide of the present invention can be used as a templatefor producing the protein of the present invention or a partial peptidethereof.

The polynucleotide of the present invention can also be used in thescreening of the present invention.

The polynucleotide of the present invention can be used for preparing atransgenic animal, a knockout animal or an animal with reducedexpression of the protein of the present invention or a protein relatedto the present invention, in accordance with conventionally knownmethods.

Since a polynucleotide comprising a nucleotide sequence complementary tothe polynucleotide of the present invention or a part thereof can detectmRNA of the protein of the present invention, for example, when used asa primer or a probe, it can be used as a primer or probe for diagnosingand/or testing an immunodeficiency disease, a cancer or an infectiousdisease or an autoimmune disease, a rejection reaction after organtransplantation, an allergic disease or an inflammatory disease. Such aprimer or probe can be labeled with an enzyme, a fluorescent material, aluminescent material or a radioisotope in accordance with conventionalmethods.

The polynucleotide of the present invention or a polynucleotidecomprising a nucleotide sequence complementary to the polynucleotide ofthe present invention or a part thereof can be used for a gene therapyfor preventing and/or treating a cancer, an immunodeficiency disease oran infectious disease or an autoimmune disease, a rejection reactionafter organ transplantation, an allergic disease or an inflammatorydisease, by introduction into cells in the living body.

The polynucleotide comprising a nucleotide sequence complementary to thepolynucleotide of the present invention or a part thereof includesso-called antisense DNA, siRNA (small interfering RNA), ribozyme and thelike.

The antisense DNA for the polynucleotide of the present invention can beproduced by inserting a part of the polynucleotide of the presentinvention (preferably DNA) into the antisense region of theaforementioned vector.

The siRNA for the polynucleotide of the present invention is adouble-stranded RNA which comprises a part of the RNA encoding theprotein of the present invention and an RNA complementary thereto. ThesiRNA can be produced by designing it based on the sequence of thepolynucleotide of the present invention, in accordance with theconventionally known method (Nature, 411: 494-498 (2001)).

The ribozyme can be produced by designing it based on the sequence ofthe polynucleotide of the present invention, in accordance with theconventionally known method (TRENDS in Molecular Medicine, 7: 221(2001)). For example, it can be produced by connecting a conventionallyknown ribozyme to a part of RNA encoding the protein of the presentinvention. The part of RNA encoding the protein of the present inventionincludes, for example, a part adjacent to a region which can be digestedby a conventionally known ribozyme (an RNA fragment). Since such anantisense DNA, siRNA or ribozyme can lower level of the protein of thepresent invention in cells, it is useful as an agent for preventingand/or treating a cancer, an immunodeficiency disease or an infectiousdisease.

When the polynucleotide of the present invention or a polynucleotidecomprising a nucleotide sequence complementary to the polynucleotide ofthe present invention or a part thereof is used as an agent forpreventing and/or treating a cancer, an immunodeficiency disease or aninfectious disease or an autoimmune disease, a rejection reaction afterorgan transplantation, an allergic disease or an inflammatory disease,the polypeptide alone, or after inserting it into an appropriate vectorsuch as a retrovirus vector or adenovirus vector, it can be administeredto human or a mammal in accordance with the usual way. Such apolynucleotide can be used directly or after making it into apharmaceutical preparation together with a carrier such as an auxiliarysubstance for accelerating its introduction into cells (e.g., liposome,HVJ liposome or the like).

According to the specification of the present invention, whennucleotides, amino acids and the like are shown by abbreviations, theyare based on the conventional abbreviations used in the field, and theirexamples include DNA (deoxyribonucleic acid), cDNA (complementarydeoxyribonucleic acid), RNA (ribonucleic acid), A (adenine), T(thymine), G (guanine), C (cytosine), Gly (glycine), Ala (alanine), Val(valine), Leu (leucine), Ile (isoleucine), Ser (serine), Thr(threonine), Cys (cysteine), Met (methionine), Glu (glutamic acid), Asp(aspartic acid), Lys (lysine), Arg (arginine), His (histidine), Phe(phenylalanine), Tyr (tyrosine), Trp (tryptophan), Pro (proline), Asn(asparagine), Gln (glutamine) and the like. Additionally, when there isa possibility of existing optical isomers regarding amino acids, theyshow L-form unless otherwise noted.

Antibody for the Protein of the Invention

According to the specification of the present invention, the antibodyfor a protein having an amino acid sequence identical to orsubstantially identical to an amino acid sequence selected from theamino acid sequences represented by SEQ ID NOs:3 to 6 or for a partialpeptide thereof (sometimes referred to as antibody of the presentinvention hereinafter) may be any antibody of a human antibody, a mouseantibody, a rat antibody, a domestic fowl antibody, a rabbit antibodyand a goat antibody, so long as it is an antibody which recognizes theprotein of the present invention or a partial peptide thereof, and itmay also be any one of their polyclonal or monoclonal antibodies,complete type or shortened type (e.g., F(ab′)₂, Fab′, Fab and Fvfragment and the like) antibodies, chimeric antibodies, humanizedantibodies or complete human antibodies. Such antibodies can be producedin accordance with the conventionally known antibody or antiserumproduction methods, using a partial peptide of the extracellular regionof the protein of the present invention as the antigen. A partialpeptide of the extracellular region of the protein can be produced inaccordance with the conventionally known protein expression andpurification methods.

The antibody of the present invention are preferably monoclonalantibodies, more preferably shortened type antibodies, chimericantibodies, humanized antibodies and complete human antibodies ofmonoclonal antibodies, and further preferably are complete humanantibodies of monoclonal antibodies.

Since the antibody for the protein of the present invention or a partialpeptide thereof can detect the protein of the present invention, it canbe used as an agent for diagnosing and/or testing an immunodeficiencydisease, a cancer or an infectious disease or an autoimmune disease, arejection reaction after organ transplantation, an allergic disease oran inflammatory disease.

Production Method and Purification Method of Protein or Polypeptide

A protein or polypeptide comprising an optional region in theextracellular region of the protein related to the present invention,which can be cited as an antagonist of the protein, a protein orpolypeptide comprising the extracellular region of the protein relatedto the present invention or a protein or polypeptide comprising theintracellular region of the same, which can be used in the screeningmethod of the present invention, and the protein of the presentinvention or a partial peptide thereon can be produced by conventionallyknown protein expression methods and purification methods or by themethods described in Examples. For example,

(1) a method for purifying and isolating them from the living body orcultured cells;

(2) a method for synthesizing peptides; and

(3) a method for producing them with the use of genetic recombinationtechniques; and the like can be cited.

The method described in (3) is industrially desirable, and as generaltechniques for that, the standard techniques described for example inMolecular Cloning (Sambrook, J., Fritsch, E. F., Maniatis, T., ColdSpring Harbor Laboratory Press) (1989) and Current Protocols inMolecular Biology (Ausubel, F. M., John Wiley & Sons, Inc. (1989)) canbe used.

An expression system (host-vector system) for producing a protein orpeptide with the use of genetic recombination techniques includesexpression systems of bacteria, yeast, insect cells and mammalian cells.

For example, when it is expressed in Escherichia coli, an expressionvector is prepared by adding an initiation codon (ATG) to the5′-terminal of a DNA coding for encoding a mature protein part;connecting the thus obtained cDNA to the downstream of an appropriatepromoter (e.g., trp promoter, lac promoter, λLPL promoter, T7 promoteror the like); and inserting it into a vector which is able to functionin E. coli cells (e.g., pBR322, pUC18, pUC19 or the like). Next, an E.coli strain (e.g., E. coli DH 1, E. coli JM 109, E. coli HB 101 strainor the like) transformed with the expression vector is cultured in anappropriate medium to obtain the objective protein or peptide from theresulting cells. Alternatively, when a bacterial signal peptide (e.g., asignal peptide of pelB or the like) is used, the objective protein orpeptide of interest can be secreted into the periplasmic space.Additionally, a fusion protein with other polypeptide can also beproduced.

Also, when it is expressed in yeast, an expression vector is prepared byconnecting a DNA encoding the protein of the present invention or apartial peptide thereof to the downstream of an appropriate promoter(e.g., PHO5 promoter, PGK promoter, GAP promoter, ADH promoter or thelike), and inserting it into a vector capable of functioning in yeast(e.g., pSH19, pSH15 or the like). Next, a yeast strain transformed withthis expression vector (e.g., Saccharomyces cerevisiae AH 22, AH 22R⁻ or20B-12, Shizosaccharomyces pombe NCYC 1913, Pichia pastoris KM 71 or thelike) is cultured in an appropriate medium to obtain the objectiveprotein or peptide.

Also, when it is expressed in an insect cell, an expression vector isprepared by connecting a DNA encoding the protein of the presentinvention or a partial peptide thereof to the downstream of anappropriate promoter (e.g., polyhedrin promoter, P10 promoter or thelike), and inserting it into a virus vector which is able to function ininsect cells. As the insect cells, for example, when the virus is AcNPV,a Barathra larva-derived established cell line (Sf cell) or the like isused. When the virus is BmNPV, a silkworm-derived established cell line(BmN cell) or the like is used. As the Sf cell, for example, Sf9 cell(ATCC CRL 1711) or Sf21 cell (In Vivo, 13: 213-217 (1977)) is used. Asthe insect, larvae of silkworm and the like are used. When an insectcell or insect is transformed, for example, it can be carried out inaccordance with the method described in Bio/Technology, 6: 47-55 (1988).

Also, when it is expressed in a mammalian cell, an expression vector isprepared by inserting a DNA encoding the protein of the presentinvention or a partial peptide thereof into the downstream of anappropriate promoter (e.g., SV40 promoter, LTR promoter, metallothioneinpromoter or the like) in an appropriate vector (e.g., a retrovirusvector, a Papillomavirus vector, a vaccinia virus vector, an SV40 systemvector or the like). Next, an appropriate mammalian cell (e.g., humanHEK 293T cell, monkey COS-1 cell, COS-7 cell, Chinese hamster CHO cell,mouse L cell, NSO cell or the like) is transformed with the thusobtained expression vector, and the resulting transformant is culturedusing an appropriate medium to express the protein of the presentinvention or a partial peptide thereof. Additionally, a fusion proteincan also be produced by ligation with a cDNA fragment encoding otherpolypeptide such as an antibody constant region (Fc moiety).

Additionally, as a method for producing a protein or peptide making useof genetic recombination techniques, a cell-free synthesis system(Sambrook J., Molecular Cloning 2nd ed. (1989)) can also be used.

The peptide synthesis method may be either a solid phase synthesismethod or a liquid phase synthesis method. The objective protein can beproduced by condensing partial peptides or amino acids which canconstitute the protein of the present invention with the remaining partsand eliminating the protecting group when the product has a protectinggroup. In the case, the condensation and elimination of protecting groupare carried out in accordance with the methods described in, forexample,

-   (i) M. Bodanszky, M. A. Ondetti, Peptide Synthesis, Interscience    Publishers, New York (1966),-   (ii) Schroeder, Luebke, The Peptide, Academic Press, New York    (1965),-   (iii) Nobuo Izumiya et al., The foundation and Experiments of    Peptide Synthesis (written in Japanese), Maruzen Co., Ltd. (1975),-   (iv) Naoaki Yajima and Shunpei Sakakibara, Biochemical    Experimentation Course 1, Chemistry of Protein IV (written in    Japanese), 205, (1977), and-   (v) edited by Naoaki Yajima, Development of Medical Supplies—a    Second Series, Volume 14, Peptide Synthesis (written in Japanese),    Hirokawa Shoten.

The protein or a partial peptide thereof obtained by the above mannercan be isolated and purified by general biochemical methods such assalting out, ion exchange chromatography, hydrophobic chromatography,affinity chromatography, reverse phase chromatography, adsorptionchromatography, chromatofocusing, isoelectric precipitation, gelfiltration, and ultrafiltration.

Additionally, it is also possible to express the protein of the presentinvention or a partial peptide thereof as a fusion protein with otherprotein or a tag (an antibody constant region, glutathioneS-transferase, protein A, FLAG tag, hexahistidine tag or the like).Since the fusion protein can be purified by affinity chromatographyand/or digestion with an appropriate protease (e.g., enterokinase,thrombin or the like), there is an advantage as it can be efficientlypurified. Method for preparing or producing polynucleotide

A polynucleotide encoding the protein related to the present inventionand a polynucleotide encoding the protein of the present invention or apartial peptide thereof can be obtained or produced by conventionallyknown preparation or production methods and purification methods, or bythe methods described in Examples. For example, it can be obtained bychemical synthesis, or by amplifying it by PCR method using a syntheticDNA primer encoding the protein of the present invention or a partialpeptide thereof, or by a hybridization method which uses a synthetic DNAencoding the protein of the present invention or a partial peptidethereof as the probe.

The human tissue or cell to be used for obtaining a polynucleotideencoding the protein of the present invention or a partial peptidethereof by the PCR method or hybridization method includes spleen, lymphnode, bone marrow, leukocyte, monocyte, B cell and the like. Byseparating mRNA from the aforementioned tissue or cell, a cDNA libraryis prepared by standard genetic recombination techniques. The objectivecDNA can be obtained by screening the library using a specific probesynthesized based on one nucleotide sequence selected from thenucleotide sequences represented by SEQ ID NOs:7 to 12. Alternatively,the objective cDNA can be amplified by synthesizing sense and antisenseprimers for amplifying the objective nucleotide sequence, which is basedon a nucleotide sequence selected from the nucleotide sequencesrepresented by SEQ ID NOs:7 to 12, and carrying out PCR using the cDNAlibrary as the template. It is desirable to carry out the PCR using anautomatic thermal cycler. The reaction can be completed in the presenceof a thermostable polymerase (e.g., Taq or the like), a template DNA andprimers, by carrying out from about 25 to 40 cycles, wherein 1 cycleincludes denaturation of DNA (e.g., 98° C., 10 to 30 seconds), annealingof primers (e.g., 56° C., 30 seconds to 1 minute) and elongationreaction in the presence of 4 kinds of substrates (dNTP) (e.g., 72° C.,30 seconds to 10 minutes), and subsequently carrying out heating at 70to 75° C. for 5 to 15 minutes. Additionally, cDNA libraries of varioushuman tissues are commercially available recently, and when they areused, the reaction can be carried out in accordance with the methoddescribed in the instructions attached thereto. The hybridization methodcan be carried out in accordance with the method described, for example,in Molecular Cloning (Sambrook, J., Fritsch, E. F., Maniatis, T., ColdSpring Harbor Laboratory Press) (1989) or Gene, 10: 63 (1980). Anecessary amount of the DNA can be obtained by introducing a vector DNAcomprising the DNA into an appropriate host and allowing it toproliferative.

Method for Preparing or Producing Antibody

Although the antibody for a protein or polypeptide comprising anoptional region in the extracellular region of the protein related tothe present invention or the antibody for the protein of the presentinvention or a partial peptide thereof may be either a polyclonalantibody or a monoclonal antibody, as long as it is an antibody whichcan recognize each of the protein or polypeptide comprising an optionalregion in the extracellular region of the protein related to the presentinvention and the protein of the present invention or a partial peptidethereof, a monoclonal antibody derived from a mammal is particularlydesirable. The monoclonal antibody derived from a mammal includes thosewhich are produced by a hybridoma and those which are produced by a hosttransformed with an expression vector comprising an antibody geneprepared by a genetic engineering technique.

An antigen-producing hybridoma can be prepared in the following mannerwith use of conventionally known techniques. Namely, a protein orpolypeptide comprising an optional region in the extracellular region ofthe protein related to the present invention, a partial peptide of theprotein of the present invention or a cell expressing the proteinrelated to the present invention or the protein of the present invention(e.g., a forced expression cell or the like) is used as a sensitizedantigen, which is used in the immunization in accordance with a generalimmunization method. The thus obtained immune cells are fused with aconventionally known parent cell by a general cell fusion method, and amonoclonal antibody producer cell is cloned by a general screeningmethod. Although the mammal to be immunized with the sensitized antigenis not particularly limited, it is desirable to select it by taking itscompatibility with the parent cell to be used in the cell fusion(myeloma cell) into consideration, and an animal of rodents, such as,mouse, rat and hamster, is generally used. Immunization of the animalwith a sensitized antigen is carried out in accordance with aconventionally known method. As the myeloma cell to be fused with theaforementioned immunized cell, various conventionally known cell strainscan be used. Cell fusion of the immunized cell with myeloma cell can becarried out in accordance with a conventionally known method such as themethod of Milstein et al. (Methods Enzymol., 73: 3-46 (1981)). The thusobtained fused cells are selected by culturing them in a generalselection medium such as HAT medium (a culture liquid containinghypoxanthine, aminopterin and thymidine). The culturing with this HATmedium is continued for generally from several days to several weeksuntil other cells than the hybridomas (un-fused cells) die out. Next,screening and cloning of a hybridoma producing an antibody which bindsto the protein of the present invention is carried out by the generallimiting dilution method. The antibody can be obtained by purifyingculture supernatant of the hybridoma obtained in this manner. Thepurification can be carried out by optionally combining generalbiochemical methods such as salting out, ion exchange chromatography andaffinity chromatography.

A polyclonal antibody can be produced by a general method in which amammal (e.g., rabbit, goat, sheep or the like) is immunized using aprotein or polypeptide comprising an optional region in theextracellular region of the protein related to the present invention orthe protein of the present invention or a partial peptide thereof as thesensitization antigen, and antiserum is recovered and purified. Thepurification can be carried out by optionally combining generalbiochemical methods such as salting out, ion exchange chromatography andaffinity chromatography.

Additionally, the antibody can also be obtained using geneticengineering techniques. Namely, mRNA is prepared from splenocyte orlymphocyte of an animal immunized by using a protein or polypeptidecomprising an optional region in the extracellular region of the proteinrelated to the present invention, a partial peptide of the protein ofthe present invention or a cell which expresses the protein related tothe present invention or the protein of the present invention (e.g.,over-expressing cell), as the sensitization antigen, or from amonoclonal antibody producer hybridoma, and a cDNA library is preparedusing this mRNA as the template. A clone producing an antibody whichreacts with the sensitization antigen is screened and the thus obtainedclone is cultured. The objective antibody can be purified from theresulting culture supernatant by optionally combining generalbiochemical methods such as salting out, ion exchange chromatography,affinity chromatography and the like. When the antibody is used as amedicine, a humanized antibody or human antibody having lowimmunogenicity is desirable. The humanized antibody can be prepared bygenetic engineering techniques using hypervariable region of theaforementioned monoclonal antibody (Method in Enzymology, 203: 99-121(199)). The human antibody can be prepared by immunizing a mouse whoseimmune system is replaced by its human counterpart (Nat. Genet., 15:146-156 (1997)).

An antibody for a protein or polypeptide comprising an optional regionin the extracellular region of the protein related to the presentinvention, as an antagonist for the protein related to the presentinvention, can be selected by the following method. Namely, an antibodyfor an activated receptor (e.g., BCR or the like) which is co-expressedwith the protein related to the present invention, or a ligand of theprotein related to the present invention is simultaneously immobilizedon a carrier such as agarose beads or a culture plate. When a cellexpressing the protein related to the present invention (e.g., B cell,monocyte, over-expressing cell or the like) is added to theaforementioned immobilized carrier or plate to carry out stimulation, anantibody to be tested is added at the same time. Whether or not itattenuates or inhibits suppression of intracellular signal transductionby the protein related to the present invention is evaluated, usingintracellular Ca²⁺ concentration, phosphorylation of intracellulartyrosine residue of the protein related to the present invention,binding of a phosphatase to the protein related to the presentinvention, phosphorylation of a signal transducer molecule such as anErk2, or produced amount of a cytokine (e.g., IL-2 or the like), as theindex.

An agonist antibody for the protein related to the present invention canbe selected by the following method. That is, an antibody to be testedis simultaneously immobilized on a carrier such as agarose beads or aculture plate. By adding a cell expressing the protein of the presentinvention (e.g., B cell, monocyte, over-expressing cell or the like) tothe aforementioned immobilized carrier or plate, whether or not it keepsor reinforces suppression of intracellular signal transduction by theprotein related to the present invention is evaluated, usingintracellular Ca²⁺ concentration, phosphorylation of intracellulartyrosine residue of the protein of the present invention, binding of aphosphatase to the protein related to the present invention,phosphorylation of a signal transducer molecule such as an Erk2, orproduced amount of a cytokine (e.g., IL-2 or the like), as the index.

Application to Medical Supplies

Since an antagonist of the protein related to the present invention or apharmaceutical composition comprising the same has an immunopotentiationactivity, it can be used for preventing and/or treating of cancers,immunodeficiency diseases or infectious diseases.

Examples of the cancers or tumors, wherein its prevention and/ortreatment can be expected by the administration of an antagonist of theprotein related to the present invention or a pharmaceutical compositioncomprising the same, include lingual cancer, gingival cancer, malignantlymphoma, malignant melanoma (melanoma), maxillary cancer, nasal cancer,nasal cavity cancer, laryngeal cancer, pharyngeal cancer, glioma,myeloma, glioma, neuroblastoma, papillary carcinoma of thyroid,follicular carcinoma of thyroid, medullary carcinoma of thyroid, primarypulmonary carcinoma, squamous cell carcinoma, adenocarcinoma, alveolarcarcinoma, large cell undifferentiated carcinoma, small cellundifferentiated carcinoma, carcinoid, testicular tumor, prostaticcancer, breast cancer (e.g., papillary adenocarcinoma, comedocarcinoma,mucous tumor, medullary carcinoma, lobular carcinoma, scirrhouscarcinosarcoma, metastatic tumor), mammary Paget disease, mammarysarcoma, bone tumor, thyroid gland cancer, gastric cancer, liver cancer,acute myelocytic leukemia, acute promyelocytic leukemia, acutemyelomonocytic leukemia, acute monocytic leukemia, acute lymphocyticleukemia, acute undifferentiated leukemia, chronic myeloid leukemia,chronic lymphocytic leukemia, adult T cell leukemia, malignant lymphoma(e.g., lymphosarcoma, reticulum cell sarcoma, Hodgkin disease or thelike), multiple myeloma, primary macroglobulinemia, infantile leukemia,esophageal carcinoma, gastric cancer, gastrocolic leiomyosarcoma,gastrointestinal malignant lymphoma, pancreas-gall bladder cancer,duodenal cancer, large bowel cancer, primary cancer of liver (e.g.,hepatocellular carcinoma, bile duct cancer or the like), hepatoblastoma,uterine intraepithelial carcinoma, cervical squamous cell carcinoma,adenocarcinoma of uterus, adenosquamous carcinoma of uterus, uterinebody adenocarcinoma, uterine carcinosarcoma, invasive hydatidiform moleof uterus, malignant chorioepithelioma of uterus, uterine malignantmelanoma, ovarian cancer, mesodermal mixed tumor, renal carcinoma, renalpelvic transitional cell carcinoma, ureteral transitional cellcarcinoma, papillary carcinoma of urinary bladder, bladder transitionalcell carcinoma, urethral squamous cell carcinoma, urethraladenocarcinoma, Wilms tumor, rhabdomyosarcoma, fibrosarcoma,osteosarcoma, chondrosarcoma, synovial sarcoma, myxosarcoma,liposarcoma, Ewing sarcoma, skin squamous cell carcinoma, skin basalcell carcinoma, skin Bowen disease, skin Paget disease, skin malignantmelanoma, malignant mesothelioma, metastatic adenocarcinoma, metastaticsquamous cell carcinoma, metastatic sarcoma, mesothelioma (e.g., pleuralmesothelioma, peritoneal mesothelioma, pericardial mesothelioma or thelike) and the like.

Examples of the immunodeficiency diseases, wherein its prevention and/ortreatment can be expected by the administration of an antagonist of theprotein related to the present invention or a pharmaceutical compositioncomprising the same, include acquired immunodeficiency syndrome (AIDS)caused by human immunodeficiency virus infection (e.g., candidaesophagitis, carinii pneumonia, toxoplasmosis, tuberculosis,Mycobacterium avium complex infection, cryptosporidiosis, Cryptococcusmeningitis, cytomegalic inclusion disease, opportunistic infection suchas progressive multifocal leuco-encephalopathy, or the like),immunodeficiency and primary immunodeficiency syndrome accompanied by aserious disease (e.g., cancer, hypoplastic anemia, leukemia,myelofibrosis, renal insufficiency, diabetes, hepatic disease or splenicdisease) and the like.

It is considered that a virus uses a coupling suppression factor ofimmune cells as a method for escaping from immunoprophylaxis of infectedhost (Journal of Experimental Medicine, 191, 11: 1987-1997 (2000)).Since viral infection is partly caused by such an escaping function ofviruses, it is considered that immune reaction of immune cells withviruses can be improved by the administration of an antagonist of theprotein related to the present invention or a pharmaceutical compositioncomprising the same.

Examples of the infectious diseases, wherein its prevention and/ortreatment can be expected by the administration of an antagonist of theprotein related to the present invention or a pharmaceutical compositioncomprising the same, include infections with human hepatitis viruses(e.g., hepatitis B, hepatitis C, hepatitis A and hepatitis E), humanretrovirus, human immunodeficiency viruses (e.g., HIV 1 and HIV 2),human T cell leukemia virus or human T lympho-tropic virus (e.g., HTLV 1and HTLV 2), type 1 or type 2 herpes simplex virus, Epstein-Barr virus,cytomegalovirus, varicella-shingles virus, human herpes viruses (e.g.,human herpes virus 6 and the like), polio virus, measles virus, rubellavirus, Japanese encephalitis virus, mumps virus, influenza virus, commoncold viruses (e.g., adenovirus, enterovirus, rhinovirus and the like), avirus which causes serious acute respiratory organ syndrome (SARS),Ebola virus, Western Nile virus and the like.

Additionally, it is considered that this is also effective forinfections with pathogenic protozoans (e.g., Trypanosoma, malariaparasite and Toxoplasma), bacteria (e.g., Mycobacterium, Salmonella andListeria), fungi (e.g., Candida) and the like.

Since the agonist of the protein related to the present invention hasimmunosuppressive activity, it can be used for preventing and/ortreating of a disease selected from autoimmune diseases, rejectionreaction after organ transplantation, allergic diseases and inflammatorydiseases.

Examples of the autoimmune disease, wherein its prevention and/ortreatment can be expected by the administration of an agonist of theprotein related to the present invention or a pharmaceutical compositioncomprising the same, include arthritis, autoimmune hepatitis, autoimmuneglomerulonephritis, autoimmune insulitis, autoimmune orchitis,autoimmune oophoritis, ulcerative colitis, Sjogren's syndrome, Crohndisease, Behcet disease, Wegner granulomatosis, hypersensitivityangiitis, periarteritis nodosa, Hashimoto disease, myxoedema, Basedowdisease, Addison disease, autoimmune hemolytic anemia, suddenthrombopenia, pernicious anemia, myasthenia gravis, demyelinatingdisease, aortitis syndrome, psoriasis, pemphigus, pemphigoid, collagendisease (e.g., systemic lupus erythematosus, rheumatoid arthritis,diffuse scleroderma, systemic progressive sclerosis, dermatomyositis,polyarteritis nodosa, rheumatic fever or the like), Guillain-Barresyndrome, polyglandular autoimmune syndrome type II, primary biliarycirrhosis, vitiligo vulgaris, type I diabetes mellitus, and the like.Additionally, a disease having high value of lupus anticoagulationfactor can be also cited. In this connection, the disease having highvalue of lupus anticoagulation factor includes, systemic lupuserythematosus, arterial thrombosis (e.g., cerebral infarction or thelike), venous thrombosis, habitual abortion, thrombopenia,anti-phospholipid antibody syndrome and the like can be exemplified.

Examples of the rejection reaction after organ transplantation, whereinits prevention and/or treatment can be expected by the administration ofan agonist of the protein related to the present invention or apharmaceutical composition comprising the same, include rejectionreaction after renal transplantation, liver transplantation, hearttransplantation and/or lung transplantation, rejection reaction by bonemarrow transplantation, graft versus host disease and the like.

Examples of the allergic disease, wherein its prevention and/ortreatment can be expected by the administration of an agonist of theprotein related to the present invention or a pharmaceutical compositioncomprising the same, include asthma, bronchial asthma, atopicdermatitis, nettle rash, allergic rhinitis (e.g., pollinosis or thelike), allergic conjunctivitis (e.g., pollinosis or the like), allergicenterogastritis, anaphylactic shock, food allergy and the like.

Examples of the inflammatory disease, wherein its prevention and/ortreatment can be expected by the administration of an agonist of theprotein related to the present invention or a pharmaceutical compositioncomprising the same, include dermatitis (e.g., contact dermatitis,atopic dermatitis and the like), colitis (e.g., ulcerative colitis,Crohn disease and the like), angiitis (e.g., Takayasu arteritis, giantcell arteritis (temporal arteritis), polyarteritis nodosa, Wegenergranuromatosis, Churg-Strauss syndrome (allergic granuromatousangiitis), allergic skin angiitis, Henoch-Schonlein purpura,hypersensitivity angiitis, angiitis syndrome, thromboangiitis obliterans(Buerger disease), nodular vasculitis and the like), arthritis (e.g.,chronic rheumatoid arthritis, rheumatoid arthritis, osteoarthritis,tuberculous arthritis, suppurative arthritis, psoriatic arthritis,internal derangement of knee joint, idiopathic osteonecrosis,osteoarthritis and the like), hepatitis (e.g., viral hepatitis,autoimmune hepatitis and the like), nephritis (e.g., acute nephritis,chronic glomerulonephritis, rapidly progressive nephritic syndrome,acute glomerulonephritis after hemolytic streptococcal infection,membranoproliferative glomerulonephritis, Goodpasture syndrome,mesangial proliferative glomerulonephritis (IgA nephropathy),interstitial nephritis and the like), gastritis (e.g., acute infectiousgastritis, allergic gastritis, chronic gastritis and the like),pancreatitis, enteritis, laryngitis, neuritis and the like.

An antagonist or agonist of the protein related to the present inventionmay be administered as an agent for concomitant use by combining withother agent for

-   (1) compensating and/or reinforcing the preventive and/or    therapeutic effect of the preventive and/or therapeutic agent of the    present invention,-   (2) improving disposition and absorption, and reducing its dose of    the preventive and/or therapeutic agent of the present invention    and/or-   (3) reducing side effects of the preventive agent or therapeutic    agent of the present invention.

The agent for concomitant use of antagonist or agonist of the proteinrelated to the present invention with other agent may be administered inthe form of a combination agent prepared by formulating both of thecomponents in one pharmaceutical preparation or may take a form for itsadministering as separate pharmaceutical preparations. When administeredas separate pharmaceutical preparations, simultaneous administration andadministration at different times are included therein. Additionally,the administration at different times may be effected by firstlyadministering the antagonist or agonist of the protein related to thepresent invention and then administering other agent, or the other agentmay be firstly administered, followed by the administration of theantagonist or agonist of the protein related to the present invention.Respective administration method may be the same or different from eachother.

The aforementioned other agent may be a low molecular compound, or maybe a high molecular protein, polypeptide, polynucleotide (DNA, RNA orgene), antisense, decoy or antibody, a vaccine or the like. Dose of theother agent can be optionally selected using the clinically used dose asthe standard. Also, blending ratio of the antagonist or agonist of theprotein related to the present invention with the other agent can beoptionally selected based on the age and body weight of the subject tobe administered, administration method, administration time, disease orsymptom to be treated or a combination thereof. For example, from 0.01to 100 parts by mass of the other agent may be used based on 1 part byweight of the antagonist or agonist of the protein related to thepresent invention. The other agent may be administered in combination ofoptional two or more species at an optional ratio. Additionally, basedon the aforementioned mechanism, not only those which have been so farfound but also those which will be found in the future are also includedin the other agent that compensates and/or reinforces the preventiveand/or therapeutic effect of the antagonist or agonist of the proteinrelated to the present invention.

The disease which is prevented and/or treated by the aforementionedagent for concomitant use is not particularly limited, and it may be anydisease in which the preventive and/or therapeutic effect of theantagonist or agonist of the protein related to the present inventioncan be compensated and/or reinforced.

In the chemotherapy and radiotherapy for cancers, a side effect ofseverely reducing proliferation of lymphocytes is unavoidable.Administration of the antagonist of the protein related to the presentinvention or a pharmaceutical composition comprising the same shows theeffect to stimulate or proliferate the reduced lymphocytes and can alsosuppress the severe side effects accompanied by the general chemotherapyto the minimum. Also, the same thing can be said on the radiotherapy.Additionally, the dose of a chemotherapeutic agent or irradiated amountof radiation can be reduced from the generally used dose or amount ofradiation, by concomitant use with the antagonist of the protein relatedto the present invention or a pharmaceutical composition comprising thesame.

The antagonist of the protein related to the present invention can beused concomitantly with a conventional chemotherapeutic agent or madeinto a combination agent therewith. As such a chemotherapeutic agent,for example, an alkylating agent, a nitrosourea agent, a metabolicantagonist, a carcinostatic antibiotic, a plant-derived alkaloid, atopoisomerase inhibitor, a hormone therapy agent, a hormone antagonist,an aromatase inhibitor, a P glycoprotein inhibitor, a platinum complexderivative, other immunotherapy agents and other antitumor agents.Additionally, it can also be used concomitantly, or made into acombination agent, with a leuko(neutro)penia treating agent, athrombopenia treating agent, an antiemetic agent or a cancer paintreating agent, as a cancer treatment auxiliary agent.

The antagonist of the protein related to the present invention can beused concomitantly, or made into a combination agent, with otherimmunopotentiation substance. Such an immunopotentiation substanceincludes, for example, various cytokines, tumor antigens and the like.The cytokine which stimulates immune reaction includes, GM-CSF, M-CSF,G-CSF, interferon-α, β or γ, IL-1, IL-2, IL-3, IL-12 and the like.Additionally, a B7 ligand derivative, a CD3 antibody, a CD28 antibodyand a CTLA-4 antibody can also increase the immune reaction.

Administration of a cancer antigen can also improve specific immunereaction of an immunocyte for a cancer cell, and can give additional orsynergistic reinforcement by its concomitant use with the antagonist ofthe protein related to the present invention. The cancer antigen can beprepared as a purified protein when its gene is clear, or as a lysate ofthe cancer cell itself when unclear. Such a cancer antigen includes, forexample, malignant melanoma MAGE-1 and MAGE-3-derived HLA-A1 and HLA-A2commitment peptides, MART-1 and gp100. Additionally, HER2/neu peptide ofbreast cancer and ovarian cancer and MUC-1 peptide of adenocarcinoma,along with NY-ESO-1 of metastatic cancer, can be also cited.

The antagonist of the protein related to the present invention can beused concomitantly, or made into a combination agent, with an antiviralagent, an antibiotic preparation, an antibacterial agent or a visceralmycosis treating agent.

The antiviral agent includes, for example, an anti-HIV agent, ananti-influenza virus agent, an anti-herpes virus agent, interferon-α orβ, and various species of immunoglobulin.

The antagonist of the protein related to the present invention can beused concomitantly, or made into a combination agent, with a vaccine ofa virus or pathogen. The vaccine includes for example, poliomyelitisvaccine, measles virus vaccine, Japanese encephalitis vaccine, BCGvaccine, triple vaccine, mumps vaccine, varicella vaccine, influenzavaccine, hepatitis A vaccine, hepatitis B vaccine and cholera vaccine.In this connection, the anti-HIV agent includes, for example, a reversetranscriptase inhibitor (e.g., AZT, ddI, 3TC, d4T or the like), aprotease inhibitor (e.g., saquinavir mesylate, ritonavir, nelfinavirmesylate, amprenavir, delavirdine mesylate, saquinavir,lopinavir/ritonavir or the like) or a CCR5 receptor antagonist. Theanti-influenza virus agent includes, for example, influenza vaccine,oseltamivir phosphate, zanamivir hydrate, amantadine hydrochloride andthe like.

The other agent for compensating and/or reinforcing the preventiveand/or therapeutic effect of the agonist of the protein related to thepresent invention for an autoimmune disease, a rejection reaction afterorgan transplantation, an allergic disease or an inflammatory diseaseincludes, for example, a steroid agent, a non-steroidalanti-inflammatory agent, an immunosuppressive agent, an anti-allergicagent (e.g., a chemical transmitter release inhibitor, anantihistaminic, a thromboxane synthase inhibitor, a thromboxaneantagonist, a Th2 cytokine inhibitor or the like), a phosphodiesteraseinhibitor (PDE4), a mediator release inhibitor and the like.

External preparations among the steroid agents includes, for example,clobetasol propionate, diflorasone acetate, fluocinonide, mometasonefuran carboxylate, betamethasone dipropionate, betamethasone butyratepropionate, betamethasone valerate, difluprednate, budesonide,diflucortolone valerate, amcinonide, halcinonide, dexamethasone,dexamethasone propionate, dexamethasone valerate, dexamethasone acetate,hydrocortisone acetate, hydrocortisone butyrate, hydrocortisone butyratepropionate, deprodone propionate, predonisolone valerate acetate,fluocinolone acetonide, beclometasone dipropionate, triamcinoloneacetonide, flumetasone pivalate, alclometasone propionate, clobetasonebutytrate, prednisolone, peclometasone propionate, fludroxycortide andthe like.

Internal preparations or injections among the steroid agents includes,for example, cortisone acetate, hydrocortisone, hydrocortisone sodiumphosphate, hydrocortisone sodium succinate, fludrocortisone acetate,prednisolone, prednisolone acetate, prednisolone sodium succinate,prednisolone butyl acetate, prednisolone sodium phosphate, halopredoneacetate, methylprednisolone, prednisolone acetate, prednisolone sodiumsuccinate, triamcinolone, triamcinolone acetate, triamcinoloneacetonide, dexamethasone, dexamethasone acetate, dexamethasone sodiumphosphate, dexamethasone palmitate, paramethasone acetate,bethamethasone and the like, and as inhalations includes, for example,bethamethasone propionate, fluticasone propionate, budesonide,flunisolide, triamcinolone, ST-126P, ciclesonide, dexamethasoneparomitionate, mometasone furancarbonate, prasterone sulfonate,deflazacort, methylprednisolone suleptanate, methylprednisolone sodiumsuccinate and the like.

The non-steroidal anti-inflammatory agent includes, for example,aspirin, loxonin, diclofenac, celecoxib, tiaprofenic acid, alminoprofen,flurbiprofen axetil, zaltoprofen, suprofen, ketoprofen, pranoprofen,fentiazac, droxicum, ibuprofen, aceclofenac, amfenac sodium, tenoxicam,oxaprozin, piroxicam, emorfazone, tolfenamic acid, indometacin farnesil,proglumetacin maleate, sulindac, mofezolac, etodolac, lonazolac calcium,ampiroxicam, mesalazine, deflazacort, nimesulide, etoricoxib, ketorolac,trometamol, palecoxib, lobenzarit disodium, auranofin, loxoprofensodium, bucillamine, actarit, piroxicam cinnamate, nabumetone,salazosulfapyridine, lormoxicam, meloxicam, diacerein, rofecoxib,valdecoxib and the like.

The basic non-steroidal anti-inflammatory agent includes, for example,tiaramide hydrochloride, tinoridine hydrochloride, epirizole, emorfazoneand the like.

The immunosuppressive agent includes, for example, azathioprine,ascomycin, everolimus, orthoclone OKT3, corticosteroid,salazosulfapyridine, ciclosporin, cyclophosphamide, sirolimus,tacrolimus hydrate, deoxyspergualin, bucillamine, prednisolone,mycophenolate mofetil, mizoribine, methylprednisolone, methotrexate,leflunomide, anti-human lymphocyte globulin and the like.

The chemical transmitter release inhibitor among anti-allergic agentsincludes, for example, sodium cromoglycate, tranilast, amlexanox,repirinast, ibudilast, pemirolast potassium, dazanolast, nedocromil,cromoglycate, israpafant and the like.

The antihistaminic among anti-allergic agents includes, for example,diphenhydramine, diphenylpyraline hydrochloride, diphenylpyralineteoclate, clemastine fumarate, dimenhydrinate, dl-chlorpheniraminemaleate, d-chlorpheniramine maleate, triprolidine hydrochloride,promethazine hydrochloride, alimemazine tartarate, isothipendylhydrochloride, homochlorcyclizine hydrochloride, hydroxyzine,cyproheptadine, levocabastine hydrochloride, astemizole, hepotastine,desloratadine, TAK-427, ZCR-2060, NIP-530, mometasone furoate,mizolastine, BP-294, andolast, auranofin, acrivastine and the like.

The thromboxane synthase inhibitor among anti-allergic agents includes,for example, ozagrel hydrochloride, imitrodust sodium and the like. Thethromboxane antagonist includes, for example, ceratrodust, lamatroban,domitroban calcium hydrate, KT-2-962 and the like. The Th2 cytokineinhibitor includes, for example, suplatotost tosilate, sonatimod, T-614,SR-31747 and the like.

The phosphodiesterase inhibitor (PDE4) includes, for example,cilomilast, roflumilast, alophyrin, atizolam, cipamfilin, rolipram,OPC-6535, ONO-6126, IC-485, AWD-12-281, CC-10004, CC-1088, KW-4490,Iirimilast, ZK-117137, YM-976, BY-61-9987, CC-7085, CDC-998, MEM-1414,ND-1251, Ba_(y) 19-8004, D-4396, PD-168787, NIK-616, SCH-351591, V-1294Aand the like.

The mediator release inhibitor includes, for example, amlexanox,ibudilast, sodium cromoglycate, dazanolast, tranilast, pemirolastpotassium, repirinast and the like.

When active component of the antagonist or agonist of the proteinrelated to the present invention is used as a medicine, it can beadministered alone or as a pharmaceutical composition by mixing it withvarious pharmacologically acceptable preparation auxiliaries. Ingeneral, it is administered according to each purpose, in the form of apharmaceutical preparation suited for its use such as oraladministration, intravenous administration, topical administration orpercutaneous administration.

Dose of such a component varies depending on the age, body weight,symptom, therapeutic effect, administration method, treating time andthe like. In general, it is orally administered within the range of from1 ng to 10000 mg, per adult per once, from once in several days, once in3 days, once in 2 days, once a day to several times, or parenterallyadministered (preferably intravenous administration) within the range offrom 1 ng to 1000 mg, per adult per once, from once in several days,once in 3 days, once in 2 days, once a day to several times, orcontinuously administered into a vein within the range of from 1 hour to24 hours a day.

As a matter of course, since the dose changes by various conditions asdescribed in the foregoing, there is a case in which an amount smallerthan the aforementioned dose is sufficient or a case in which itsadministration of exceeding the range is necessary.

When a concomitant preparation of the antagonist or agonist of theprotein related to the present invention with ,other agent isadministered, it is used as solid preparations for internal use orsolutions for internal use for use in its oral administration, or asinjections, external preparations, suppositories, eye drops, inhalationsor the like for use in its parenteral administration.

The solid preparations for internal use for use in the oraladministration includes tablets, pills, capsules, powders, granules andthe like. Hard capsules and soft capsules are included in the capsules.Also, the tablets include sublingual tablets, buccal patch tablets,buccal quick disintegrating tablets and the like.

In such solid preparation for internal use, one or two or more of activesubstances are used as such or after making into a pharmaceuticalpreparation in accordance with the usual way by mixing them with anexcipient (e.g., lactose, mannitol, glucose, microcrystalline cellulose,starch or the like), a binder (e.g., hydroxypropyl cellulose, polyvinylpyrrolidone, aluminum magnesium metasilicate or the like), adisintegrator (e.g., calcium cellulose glycolate or the like), alubricant (e.g., magnesium stearate or the like), a stabilizer, asolubilizing agent (e.g., glutamic acid, aspartic acid or the like) orthe like. Additionally, these may be coated with a coating agent (e.g.,sucrose, gelatin, hydroxypropylcellulose, hydroxypropylmethyl-cellulosephthalate or the like) or coated with two or more layers, if necessary.Further, capsules of the absorbable substance such gelatin is alsoincluded therein.

Sublingual tablets are produced in accordance with a conventionallyknown method. For example, these are used after making one or two ormore of active substances into a pharmaceutical preparation inaccordance with the usual way by mixing them with an excipient (e.g.,lactose, mannitol, glucose, microcrystalline cellulose, colloidalsilica, starch or the like), a binder (e.g., hydroxypropyl cellulose,polyvinyl pyrrolidone, aluminum magnesium metasilicate or the like), adisintegrator (e.g., starch, L-hydroxypropylcellulose,carboxymethylcellulose, croscarmellose sodium, calcium celluloseglycolate or the like), a lubricant (e.g., magnesium stearate or thelike), a swelling agent (e.g., hydroxypropylcellulose,hydroxypropylmethylcellulose, carbopol, carboxymethylcellulose,polyvinyl alcohol, xanthan gum, guar gum or the like), a swellingauxiliary (e.g., glucose, fructose, mannitol, xylitol, erythritol,maltose, trehalose, a phosphate, a citrate, a silicate, glycine,glutamic acid, arginine or the like), a stabilizer, a solubilizing agent(e.g., polyethylene glycol, propylene glycol, glutamic acid, asparticacid or the like), a flavor (e.g., orange, strawberry, mint, lemon,vanilla or the like) or the like. Also, these may be coated with acoating agent (e.g., sucrose, gelatin, hydroxypropylcellulose,hydroxypropylmethylcellulose phthalate or the like) or coated with twoor more layers, if necessary. Additionally, generally used additiveagents such as an antiseptic, an antioxidant, a coloring agent, asweetener and the like can also be added thereto, if necesssary. Buccalpatch tablets are prepared in accordance with a conventionally knownmethod. For example, these are used after making one or two or more ofactive substances into a pharmaceutical preparation in accordance withthe usual way by mixing them with an excipient (e.g., lactose, mannitol,glucose, microcrystalline cellulose, colloidal silica, starch or thelike), a binder (e.g., hydroxypropyl-cellulose, polyvinyl pyrrolidone,aluminum magnesium metasilicate or the like), a disintegrator (e.g.,starch, L-hydroxypropylcellulose, carboxymethylcellulose, croscarmellosesodium, calcium cellulose glycolate or the like), a lubricant (e.g.,magnesium stearate or the like), an adhesive agent (e.g.,hydroxypropylcellulose, hydroxypropylmethylcellulose, carbopol,carboxymethylcellulose, polyvinyl alcohol, xanthan gum, guar gum or thelike), an adhesion auxiliary (e.g., glucose, fructose, mannitol,xylitol, erythritol, maltose, trehalose, a phosphate, a citrate, asilicate, glycine, glutamic acid, arginine or the like), a stabilizer, asolubilizing agent (e.g., polyethylene glycol, propylene glycol,glutamic acid, aspartic acid or the like), a flavor (e.g., orange,strawberry, mint, lemon, vanilla or the like) and the like. Also, thesemay be coated with a coating agent (e.g., sucrose, gelatin,hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate or thelike) or coated with two or more layers, if necessary. Additionally,generally used additive agents such as an antiseptic, an antioxidant, acoloring agent, a sweetener and the like can also be added thereto, ifnecessary. Buccal quick disintegrating tablets are prepared inaccordance with a conventionally known method. For example, one or moreof active substances are used as such or after making them into apharmaceutical preparation in accordance with the usual way by mixingthe active substances, prepared in advance by coating the bulk orgranulated bulk particles with an appropriate coating agent (e.g., ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, anacrylic acid methacrylic acid copolymer or the like) and a plasticizer(e.g., polyethylene glycol, triethyl citrate or the like), with anexcipient (e.g., lactose, mannitol, glucose, microcrystalline cellulose,colloidal silica, starch or the like), a binder (e.g.,hydroxypropyl-cellulose, polyvinyl pyrrolidone, aluminum magnesiummetasilicate or the like), a disintegrator (e.g., starch,L-hydroxypropylcellulose, carboxymethylcellulose, croscarmellose sodium,calcium cellulose glycolate or the like), a lubricant (e.g., magnesiumstearate or the like), a dispersion auxiliary (e.g., glucose, fructose,mannitol, xylitol, erythritol, maltose, trehalose, a phosphate, acitrate, a silicate, glycine, glutamic acid, arginine or the like), astabilizer, a solubilizing agent (e.g., polyethylene glycol, propyleneglycol, glutamic acid, aspartic acid or the like), a flavor (e.g.,orange, strawberry, mint, lemon, vanilla or the like) or the like. Also,these may be coated with a coating agent (e.g., sucrose, gelatin,hydroxypropyl-cellulose, hydroxypropylmethylcellulose phthalate or thelike) or coated with two or more layers, if necessary. Additionally,generally used additive agents such as an antiseptic, an antioxidant, acoloring agent, a sweetener and the like can also be added thereto, ifnecessary. The internal solutions for the oral administration includepharmaceutically acceptable waters, suspensions, emulsions, syrups,elixirs and the like. In such solutions, one or two or more of activesubstances are dissolved, suspended or emulsified in a generally useddiluent (e.g., purified water, ethanol, a mixed liquid thereof or thelike). Additionally, this solutions may contain wetting agents,suspending agents, emulsifying agents, sweeteners, flavors, aromatics,preservatives, buffer agents or the like.

For example, the external preparations for the parenteral administrationinclude ointments, gels, creams, fomentations, patches, liniments,aerosols, inhalations, sprays, aerosols, eye drops, nasal drops and thelike. These contain one or two or more of active substances and areprepared by a conventionally known method or a generally usedprescription.

Ointments are produced by a conventionally known or generally usedmethod. For example, they are prepared by mixing or melting one or twoor more of active substances in a base. The ointment base is selectedfrom those which are conventionally known or generally used. Forexample, those which are selected from a higher fatty acid or higherfatty acid ester (e.g., adipic acid, myristic acid, palmitic acid,stearic acid, oleic acid, adipic acid ester, myristic acid ester,palmitic acid ester, stearic acid ester, oleic acid ester or the like),waxes (e.g., yellow beeswax, spermaceti wax, ceresin or the like), asurfactant (e.g., polyoxyethylene alkyl ether phosphoric acid ester orthe like), a higher alcohol (e.g., cetanol, stearyl alcohol, cetostearylalcohol or the like), a silicon oil (e.g., dimethylpolysiloxane or thelike), hydrocarbons (e.g., hydrophilic petrolatum, white petrolatum,purified lanolin, liquid paraffin or the like), glycols (e.g., ethyleneglycol, diethylene glycol, propylene glycol, polyethylene glycol,macrogol or the like), a plant oil (e.g., castor oil, olive oil, sesameoil, turpentine oil or the like), an animal oil (e.g., mink oil, yolkoil, squalane, squalene or the like), water, an absorption acceleratorand a rash preventing agent are used alone or as a mixture of two ormore. Additionally, they may contain moisture keeping agents,preservatives, stabilizers, antioxidants, flavors or the like.

Gels are produced by a conventionally known or generally used method.For example, they are prepared by melting one or two or more of activesubstances in a base. The gel base is selected from those which areconventionally known or generally used. For example, those which areselected from a lower alcohol (e.g., ethanol, isopropyl alcohol or thelike), a gelling agent (e.g., carboxymethyl cellulose, hydroxyethylcellulose, hydroxypropyl cellulose, ethyl cellulose or the like), aneutralizing agent (e.g., triethanolamine, diisopropanolamine or thelike), a surfactant (e.g., polyethylene glycol monostearate or thelike), gums , water, an absorption accelerator and a rash preventingagent are used alone or as a mixture of two or more. Additionally, theymay contain preservatives, antioxidants, flavors or the like.

Creams are produced by a conventionally known or generally used method.For example, they are prepared by melting or emulsifying one or two ormore of active substances in a base. The cream base is selected fromthose which are conventionally known or generally used. For example,those which are selected from a higher fatty acid ester, a loweralcohol, hydrocarbons, a polyhydric alcohol (e.g., propylene glycol,1,3-butylene glycol or the like), a higher alcohol (e.g.,2-hexyldecanol, cetanol or the like), an emulsifying agent (e.g.,polyoxyethylene alkyl ethers, fatty acid esters or the like), water, anabsorption accelerator and a rash preventing agent are used alone or asa mixture of two or more. Additionally, they may contain preservatives,antioxidants, flavors or the like.

Fomentations are produced by a conventionally known or generally usedmethod. For example, they are produced by melting one or two or more ofactive substances in a base, and spreading and coating it on a supportas a kneaded material. The fomentation base is selected from those whichare conventionally known or generally used. For example, those which areselected from a thickener (e.g., polyacrylic acid, polyvinylpyrrolidone, acacia, starch, gelatin, methyl cellulose or the like), awetting agent (e.g., urea, glycerol, propylene glycol or the like), abulking agent (e.g., kaolin, zinc oxide, talc, calcium, magnesium or thelike), water, a solubilizing agent, a tackifier and a rash preventingagent are used alone or as a mixture of two or more. Additionally, theymay contain preservatives, antioxidants, flavors or the like.

Patches are produced by a conventionally known or generally used method.For example, they are prepared by melting one or two or more of activesubstances in a base, and spreading and coating it on a support. Thepatch base is selected from those which are conventionally known orgenerally used. For example, those which are selecting from a highmolecular base, a fat, a higher fatty acid, a tackifier and a rashpreventing agent are used alone or as a mixture of two or more.Additionally, preservatives, antioxidants, flavors or the like may alsobe contained therein.

Liniments are produced by a conventionally known or generally usedmethod. For example, they are prepared by dissolving, suspending oremulsifying one or two or more of active substances in one or two ormore of materials selected from water, an alcohol (e.g., ethanol,polyethylene glycol or the like), a higher fatty acid, glycerol, soap,an emulsifying agent and a suspending agent. Additionally, they maycontain preservatives, antioxidants, flavors or the like.

The aerosols, inhalations and sprays may contain a stabilizer such assodium hydrogen sulfite and a buffer agent which provides tonicity,which is a tonicity agent such as sodium chloride, sodium citrate orcitric acid, in addition to the generally used diluent. Productionmethods of sprays are described in detail, for example, in U.S. Pat.Nos. 2,868,691 and 3,095,355.

The injections for parenteral administration include all injections andalso include drip infusions. For example, they include intramuscularinjections, subcutaneous injections, intradermal injections,intraarterial injections, intravenous injections, intraperitonealinjections, spinal injections, intravenous drip infusions and the like.

The injections for parenteral administration include solutions,suspensions, emulsions and solid injections which are used by dissolvingor suspending in a solvent when used. The injections are used bydissolving, suspending or emulsifying one or two or more of activesubstances in a solvent. As the solvent, for example, distilled waterfor injection, physiological saline, plant oil, alcohols such aspropylene glycol, polyethylene glycol or ethanol, and the like and acombination therewith are used. Additionally, such injections maycontain a stabilizing agent, a solubilizing agent (e.g., glutamic acid,aspartic acid, Polysorbate 80 (registered trademark) or the like), asuspending agent, an emulsifying agent, a soothing agent, a bufferagent, a preservative or the like. These are sterilized in the finalprocess or prepared by an aseptic operation. Alternatively, it ispossible to produce an aseptic solid preparation such as a freeze-driedproduct, and to use it by dissolving in sterilized or aseptic distilledwater for injection or other solvent prior to its use.

The eye drops for parenteral administration include ophthalmicsolutions, suspension type ophthalmic solutions, emulsion typeophthalmic solutions, ophthalmic solutions of a dissolving type whenused and eye ointments.

These eye drops are produced in accordance with conventionally knownmethods. For example, these are used by dissolving, suspending oremulsifying one or two or more of active substances in a solvent. As thesolvent of eye drops, for example, aseptic purified water, physiologicalsaline, other aqueous solvent or non-aqueous solvent for injection(e.g., a plant oil or the like) and the like and a combination thereofare used. The eye drops may contain tonicity agents (e.g., sodiumchloride, concentrated glycerol and the like), buffer agents (e.g.,sodium phosphate or sodium acetate and the like), surfactants (e.g,Polysorbate 80 (registered trademark), Polyoxyl 40 stearate, forexample, polyoxyethylene hydrogenated castor oil and the like),stabilizers (e.g., sodium citrate or sodium edetate and the like),antiseptics (e.g., benzalkonium chloride or paraben and the like), byoptionally selecting them, if necessary. These are sterilized in thefinal process or prepared by an aseptic operation. Alternatively, it ispossible to produce an aseptic solid preparation such as a freeze-driedproduct, and to use it by dissolving in sterilized or aseptic sterilizedpurified water or other solvent prior to its use.

The inhalations for parenteral administration include aerosols, powdersfor inhalation or solutions for inhalation, and the solutions forinhalation may be such a form that they are used by dissolving orsuspending in water or other appropriate solvent at the time of theiruse.

These inhalations are produced in accordance with conventionally knownmethods.

For example, in the case of solutions for inhalation, they are preparedby optionally selecting antiseptics (e.g., benzalkonium chloride orparaben and the like), coloring agents, buffer agents (e.g., sodiumphosphate or sodium acetate and the like), tonicity agents (e.g., sodiumchloride or concentrated glycerol and the like), thickeners (e.g.,carboxy vinyl polymer and the like), absorption promoters and the like,if necessary.

In the case of powders for inhalation, they are prepared by optionallyselecting lubricants (e.g., stearic acid and salts thereof and thelike), binders (e.g., starch, dextrin and the like), excipients (e.g.,lactose or cellulose and the like), coloring agents, antiseptics (e.g.,benzalkonium chloride or paraben and the like), absorption acceleratingagents and the like, if necessary.

When the solutions for inhalation are administered, a sprayer (e.g., anatomizer or nebulizer) is generally used, and an inhaler for powderagents is generally used when the powders for inhalation areadministered.

Other compositions for parenteral administration include suppositoriesfor rectal administration, pessaries for vaginal administration and thelike, which comprise one or two or more active substances and areprescribed in the usual way.

EXAMPLES

The present invention is described below in more detail with referenceto examples, but the present invention is not limited thereto.

Example 1 Expression Profile of Human BIR1

In order to examine expression of BIR1 mRNA in human normal tissues,blood cells and cell strains, BIR1-specific primers were designed, andPCR was carried out using TaKaRa Ex Taq (manufactured by TaKaRa).Sequences of the primers used therein are shown below.

5′-GAACAGGCTCCTCTTCTGGAG-3′ (SEQ ID NO: 13) 5′-GGTTCACCTTTTCCATCCTGG-3′(SEQ ID NO: 14)

The PCR was carried out by firstly keeping at 96° C. for 1 minute,subsequently repeating 35 cycles of a temperature operation of 98° C.for 10 seconds, 56° C. for 30 seconds and 72° C. for 30 seconds, andfinally keeping at 72° C. for 10 minutes.

Human MTC Panel I, Human MTC Panel II, Human Immune System MTC Panel andHuman Blood Fractions MTC Panel (manufactured by BD Clontech) were usedin the expression analysis of human normal tissues and blood cells, andcDNA prepared from total RNA by reverse transcription reaction inaccordance with the usual way was used in the expression analysis ofhuman cell lines. Each of the PCR products was subjected to agarose gelelectrophoresis, and then the gel was stained with ethidium bromide toobtain image data by BioDoc-It System (manufactured by UVP). The resultsare shown in FIG. 1.

BIR1 was expressed at high level in immune system tissues such as spleenand lymph node and in CD14-positive cells (monocytes) and CD19-positivecells (B cells). Additionally, in the case of human cell lines,expression of BIR1 was found in K562 and HL-60 which have the monocytedifferentiation ability, monocyte system cell strains U937 and THP-1,and B cell strains Raji, CCRF-SB and FLEB-14-14.

Example 2 Expression of Human BIR1 by Various Inflammation Stimuli

Expression of BIR1 by various inflammation stimuli was examined usinghuman monocyte system cell strains. At a density of 1×10⁶ cells/2 mL,each of THP-1 cells and U937 cells were stimulated withlipopolysaccharide (LPS) (1 μg/mL), phorbol myristate acetate (PMA) (100ng/mL), IFN-γ (100 ng/mL), TNF-α (10 ng/mL) or LPS+IFN-γ (1 μg/mL+100ng/mL), for 1, 3, 8, 24, 48 or 120 hours, and the total RNA wasrecovered. Amount of RNA of BIR1 was determined by ABI PRISM 7000Sequence Detection System (manufactured by Applied Biosystems) usingBIR1-specific primers; 5′-CACAGCCATGGAAGTTGGAATC-3′ (SEQ ID NO:15) and5′-GAGTGTTTGGCCTCATCTTGG-3′ (SEQ ID NO:16) and QuantiTect SYBR GreenRT-PCR Kit (manufactured by QIAGEN). The PCR was carried out by firstlykeeping at 50° C. for 30 minutes and then at 95° C. for 15 minutes.Next, a temperature operation of 94° C. for 10 seconds, 56° C. for 30seconds and 72° C. for 1 minute was repeated 45 times. As a result, asshown in FIG. 2, gene expression of BIR1 was increased in 8 to 48 hoursafter the stimulation other than TNF-α in THP-1 cells, and was increasedin 24 to 48 hours after any of the stimuli in U937 cells.

Example 3 Expression of BIR1 in Autoimmune Disease Patient-Derived BloodCells

Expression of BIR1 in autoimmune disease patient-derived various bloodcells were detected using Autoimmune Disease Profiling Array(manufactured by BD Clontech). A BIR1-specific probe labeled with[α-³²P] dCTP (manufactured by Perkin Elmer) was prepared using RandomPrimer DNA Labeling Kit Ver. 2 (manufactured by TaKaRa), using a humanBIR1 partial length cDNA fragment as the template. The partial lengthcDNA fragment used therein is shown in SEQ ID NO:17.

The pre-hybridization, hybridization and washing operations after theprobe preparation were carried out in accordance with the instructionsattached thereto. By obtaining image data using BAS 2000 (manufacturedby FUJIFILM), PSL (photo-stimulated luminescence; proportional toradiation dose) value of each dot was calculated from the image datausing the Image Analyzer II of BAStation Ver. 2.21, and the expressedamount of BIR1 in each sample was numerically expressed. As thestatistical analysis, Wilcoxon (Mann-Whitney) test was carried out onhealthy persons. The results are shown in FIG. 3.

In comparison with healthy persons, expression of BIR1 was significantlyincreased in monocytes of lupus anticoagulation factor patients and Bcells of the patients of articular rheumatism, multiple sclerosis andTakayasu arteritis.

Example 4 Identification of Human BIR1 Splicing Variants

PCR was carried out using human CD14-positive cell (monocyte)- andCD19-positive cell (B cell)-derived cDNA (Human Blood Fractions MTCPanel) (manufactured by BD Clontech) as the template and using TaKaRa LATaq (manufactured by TaKaRa). The designed primers specific to humanBIR1 are shown below.

5′-ATGTGGAGCCATTTGAACAGGCTCCTC-3′ (SEQ ID NO: 18)5′-TCAGAAGTTGAGTTCAGAATAGAC-3′ (SEQ ID NO: 19)

The PCR was carried out by firstly keeping at 96° C. for 1 minute,subsequently repeating 35 cycles of a temperature operation of 98° C.for 10 seconds, 56° C. for 30 seconds and 72° C. for 1 minute and 30seconds, and finally keeping at 72° C. for 10 minutes. The PCR productswere subjected to agarose gel electrophoresis and then isolated andsubcloned into T/A vector. In accordance with the instructions attachedto BigDye Terminator v3.1 Cycle Sequencing Kit (manufactured by AppliedBiosystems), the sequence of each cDNA fragment was determined by ABIPRISM 3100 Genetic Analyzer (manufactured by Applied Biosystems).

As a result, in addition to the known isoform (two immunoglobulin (Ig)domains are present; BIR1L) (SEQ ID NOs:1 and 2), an isoform in whichthe Ig domain at the C-terminal side is deleted (BIR1S1) (SEQ ID NO:3),an isoform in which the Ig domain at the N-terminal side is deleted(BIR1S2) (SEQ ID NO:4), an isoform in which both of the Ig domains aredeleted (BIR1ΔIg) (SEQ ID NO:5) and an isoform in which a part of theintracellular region of BIR1S1 is deleted (BIR1ΔCyt) (SEQ ID NO:6) werenewly identified. The Ig domain at the N-terminal side is a part whichcorresponds to the amino acid numbers 41 to 122 of the amino acidsequence described in SEQ ID NO:1, and the Ig domain at the C-terminalside is a part which corresponds to the amino acid numbers 138 to 203 ofthe amino acid sequence represented by SEQ ID N0:1.

Example 5 Preparation of Cell Stably Expressing Mouse FcγRIIB-Human BIR1Chimeric Protein

Examination was carried out whether or not BIR1 transmits an inhibitorysignal. The following materials were prepared in accordance with themethods reported in Proc. Natl. Acad. Sci. U.S.A., 98: 13866-13871(2001), J. Immunol., 162: 3168-3175 (1999) and the like. An FcR chimericprotein expression vector was constructed by inserting a DNA encoding anFcR-hBIR1 (wt) chimeric protein (SEQ ID NO:20), which was prepared byconnecting the intracellular region of BIR1 (amino acid numbers 251 to343 of SEQ ID NO:1) to the C-terminal side of the extracellular region,transmembrane region and 6 intracellular region amino acids (amino acidnumbers 1 to 252) of mouse FcγRIIB (FcR), into downstream of the β-actinpromoter. Also, an expression vector of a mutant (FcR-hBIR1 (YWF)) (SEQID NO:21), in which all of the 6 tyrosine residues of the intracellularregion of BIR1 were replaced by phenylalanine residues, was preparedusing QuickChange Multi Site-Directed Mutagenesis Kit (manufactured byStratagene). These FcR chimeric protein expression vectors and anexpression vector for an FcR chimeric protein of a known inhibitoryreceptor human KIR2DL3 (FcR-hKIR2DL3) (SEQ ID NO:22) were introducedinto a mouse B cell strain A20IIA1.6 (a mouse FcγRIIB-deficient cellstrain) (J. Immunol., 136: 348-356 (1986)) using Gene Pulser XcellElectroporation System (manufactured by BIO-Rad). Each of the introducedcells was stained with an FITC-labeled rat anti-mouse CD16/CD32 antibody(2.4G2) (manufactured by BD Pharmingen), and then expression level ofthe FcR chimeric protein was examined by FACSCalibur (manufactured by BDBiosciences).

As shown in FIG. 4, these were cells which stably express the FcRchimeric protein at almost the same level.

Example 6 Inhibition of Intracellular Ca²⁺ Concentration Increase byHuman BIR1 Via B Cell Receptor

The cell which stably expresses FcR chimeric protein was allowed toincorporate Fura2-AM (final concentration: 5 μmol/l) (manufactured byWako Pure Chemical Industries) at 37° C. for 30 minutes in the presenceof 2.5 mmol/L of probenecid. After removing Fura2-AM, it was allowed tostand at 37° C. for 30 minutes in HEPES/Hanks' buffer containing 2.5mmol/L of probenecid. After discarding the supernatant bycentrifugation, the cells are suspended in HEPES/Hanks' buffercontaining 2.5 mmol/L of probenecid at a density of 5×10⁶ cells/mL andinoculated into a 96 well plate for Ca²⁺ measurement at 100 μL/well. Ina test in the absence of extracellular Ca²⁺, the same operation wascarried out using HEPES/Hanks' buffer containing 1 mmol/L EGTA/2.5mmol/L probenecid prepared using Ca²⁺-free Hanks' buffer. After standingat room temperature for 30 minutes, a fluorescence intensity ratio of340 nm/380 nm was measured using Spectrofluorometer FDSS-4000(manufactured by Hamamatsu Photonics), and a change in the intracellularCa²⁺ concentration was detected. After 30 seconds of the commencement ofmeasurement, F(ab′)₂ (33 μg/mL) of or intact (49.5 μg/mL) rabbitanti-mouse IgG (H+L) antibody (manufactured by Zymed) was added theretoin 10 μL/well portions.

As shown in FIG. 5, it was found that, when the BIR1 chimeric proteinwas crosslinked with BCR by the addition of the intact antibody, theBIR1 chimeric protein inhibited increase of intracellular Ca²⁺concentration, via BCR. On the other hand, increase of intracellularCa²⁺ concentration was not inhibited in the mutant in whichintracellular tyrosine residues of BIR1 were replaced by phenylalanineresidues. Additionally, the intracellular Ca²⁺ concentration increaseinhibition pattern of BIR1 was different from that of the positivecontrol KIR2DL3. When intracellular Ca²⁺ concentration increaseinhibition patters of BIR1 and KIR2DL3 were compared in the absence ofextracellular Ca²⁺, KIR2DL3 completely inhibited increase ofintracellular Ca²⁺ concentration in the absence of extracellular Ca²⁺,while BIR1 was unable to inhibit. BIR1 showed similar results of a knowninhibitory receptor FcγRIIB (Cell, 90: 293-301 (1997)).

Based on the above results, it was shown that the BIR1 as a proteinrelated to the present invention functions as an immunosuppressivereceptor.

Example 7 Phosphorylation of Human BIR1 Intracellular Tyrosine Residueand Identification of Recruited Phosphatase, in Inhibitory SignalTransduction

Examination was carried out on whether or not intracellular tyrosineresidue is phosphorylated and a phosphatase is recruited when BIR1 isactivated. With 30 μg/mL of F(ab′)₂ of rabbit anti-mouse IgG (H+L)antibody or 45 μg/mL of intact antibody thereof(manufactured by Zymed),3×10⁷ cells of the FcR chimeric protein-stably expressing cell suspendedin Ca²⁺-containing HEPES/Hanks' buffer were stimulated at 37° C. for 3minutes. Thereafter, the cells were lysed with a lysis buffer (1% NP-40,50 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl, 50 mmol/L NaF, 10%glycerol, 1 mmol/L Na₃VO₄, 1 mol/L PMSF, proteinnase inhibitor cocktailtablet (manufactured by Roche Diagnostics)). The cell lysate was allowedto react with Protein A/G PLUS-Agarose (manufactured by Santacruz) towhich a rat anti-mouse CD16/CD32 antibody (2.4G2) (manufactured by BDPharmingen) is bound, at 4° C. for 5 hours or more. The FcR chimericprotein and binding molecules thereof are immuno-precipitated and thenWestern blotting was carried out in the usual way. The membrane wasallowed to react with phosphotyrosine (4G10) (manufactured by Upstate)and primary antibodies for SHP-1, SHP-2 and SHIP-1 (manufactured bySantacruz), followed by the reaction with a horse radish peroxidase(HRP)-labeled secondary antibody (manufactured by Santacruz) to detectthe bands by an ECL detection system (manufactured by AmershamBiosciences).

As a result, as shown in FIG. 6, intracellular tyrosine residue of BIR1chimeric protein was phosphorylated when the BIR1 chimeric protein wascrosslinked with BCR caused by the addition of the intact antibody.Additionally, SHP-1, SHP-2 and SHIP-1 were recruited into theintracellular region of BIR1 chimeric protein at that time. On the otherhand, the phosphatase was not recruited in the mutant in whichintracellular tyrosine residue of BIR1 was replaced by phenylalanineresidue.

Example 8 Inhibition of Phosphorylation of Erk2 by Human BIR1

Examination was carried out whether or not BIR1 inhibits phosphorylationof the downstream signal transduction molecule via BCR. With 30 μg/mL ofF(ab′)₂ of rabbit anti-mouse IgG (H+L) antibody or 45 μg/mL of intactantibody thereof (manufactured by Zymed), 6×10⁶ cells which stablyexpress FcR chimeric protein suspended in Ca²⁺-containing HEPES/Hanks'buffer were stimulated at 37° C. for 3 minutes. A cell lysate (500ng/μl) was prepared using a lysing solution (Cell Lysis Kit;manufactured by BIO-RAD). Phosphorylation of each signal transductionmolecule was determined using Bio-Plex Phospho 7-Plex Assay(manufactured by BIO-RAD).

As a result, as shown in FIG. 7, BIR1 chimeric protein inhibitedBCR-mediated phosphorylation of Erk2 when the BIR1 chimeric protein wascrosslinked with BCR by the addition of the intact antibody, similar tothe case of KIR2DL3. On the other hand, the mutant in whichintracellular tyrosine residue of BIR1 was replaced by phenylalanineresidue did not inhibit phosphorylation of Erk2.

Example 9 Inhibition of Interleukin 2 (IL-2) Production by Human BIR1

Examination carried out on whether or not BIR1 inhibits BCR-mediatedIL-2 production. After inoculation into 96 well plates, 1×10⁵ cellswhich stably express the FcR chimeric protein were stimulated with 10μg/mL of F(ab′)₂ of rabbit anti-mouse IgG (H+L) antibody or 5 pg/mL ofintact antibody thereof(manufactured by Zymed) at 37° C. for 24 hours.The amount of IL-2 in each culture supernatant was measured usingQuantikine Immunoassay Mouse IL-2 ELISA Kit (manufactured by R & DSystems). As a result, as shown in FIG. 8, BIR1 chimeric proteininhibited BCR-mediated IL-2 production when the BIR1 chimeric proteinwas crosslinked with BCR by the addition of the intact antibody. On theother hand, the mutant in which intracellular tyrosine residue of BIR1was replaced by phenylalanine residue did not inhibit IL-2 production.Additionally, as has been previously reported, KIR2DL3 also inhibitedIL-2 production.

Example 10 Identification of ITIM Domain of Human BIR1 and BindingPhosphatase

The ITIM domain which is a sequence important for BIR1 in transmittinginhibitory signal and the binding phosphatase were identified. Peptides(Y3: Biotin-HSQELQ³¹³YATPVF (SEQ ID NO:23), Y5: Biotin-DSYKSG³³⁶YVYSEL(SEQ ID NO:24), Y6: Biotin-YKSGYV³³⁸YSELNF (SEQ ID NO:25)) containingintracellular tyrosine of BIR1 (SEQ ID NO:1) and their correspondingphosphorylated peptides (pY3: Biotin-HSQELQ³¹³(pY)ATPVF (SEQ ID NO:26),pY5: Biotin-DSYKSG³³⁶(pY)VYSEL (SEQ ID NO:27), pY6:Biotin-YKSGYV³³⁸(pY)SELNF (SEQ ID NO:28)) were synthesized (consigned toSigma Genosys). A peptide containing the ITIM sequence of mouse. PIR-Band its phosphorylated peptide (Y3: Biotin-ESQDVT⁷⁹⁴YAQLCS (SEQ IDNO:29) and pY3: Biotin-ESQDVT⁷⁹⁴(pY)AQLCS (SEQ ID NO:30)) weresynthesized as positive controls. These peptides were connected toProtein A/G PLUS-Agarose (manufactured by Santacruz) via an anti-biotinantibody (manufactured by Sigma) and then allowed to react with a lysateof A20IIA1.6 cell at 4° C. for 2 hours. After immunoprecipitation,western blotting was carried out in the usual way. The membrane wasallowed to react with primary antibodies for SHP-1, SHP-2 and SHIP-1(manufactured by Santacruz), and then allowed to react with an(HRP)-labeled secondary antibody (manufactured by Santacruz), and thebands were detected using an ECL plus Western Blotting Detection System(manufactured by Amersham Biosciences).

As a result, as shown in FIG. 9, pY3 of BIR1 bound to SHP-1 and SHP-2,and pY6 bonded to SHP-1, SHP-2 and SHIP-1. On the other hand, pY5 didnot bind to these phosphatases. Although a sequence which coincides withthe consensus sequence of the conventional ITIM (I/V/L/SXYXXL/V) is notpresent in the intracellular region of BIR1, it was found that the 313thand 338th tyrosine residues of human BIR1 function as new ITIM.

Example 12 Preparation of Anti-Human BIR1 Antibody (1) AntigenSensitization Using Solubilized Human BIR1L/Fc Chimeric Protein

After mixing with TiterMax adjuvant (manufactured by Sigma), 60 μgportion of solubilized human BIR1L/Fc chimeric protein was administeredinto the intraperitoneal cavity of a BALB/c mouse. Two weeks after theinitial administration, the antigen (60 μg) was mixed with TiterMaxadjuvant and administered into the intraperitoneal cavity of the mouse.After about 10 days after the booster, the antigen (40 μg) was finallyadministered into the intraperitoneal cavity of the mouse. Three daysthereafter, the spleen was harvested from the mouse.

(2) Preparation of Anti-Human BIR1 Monoclonal Antibody

Lymphocytes were separated from the spleen obtained in theaforementioned (1) and mixed with mouse myeloma P3U1 at a ratio of about4:1 to carry out cell fusion using polyethylene glycol. Hybridomas wereselected using RPMI 1640/15% FCS/HAT medium, and screening of hybridomasproducing the objective antibody was carried out by an ELISA using thesoluble human BIR1L/Fc chimeric protein and a flow cytometry using a CHOcell which stably express human BIR1L. Positive hybridomas were clonedby limiting dilution to obtain an anti-human BIR1 monoclonal antibodyproducer hybridoma. The hybridoma obtained by the manner was inoculatedinto the intraperitoneal cavity of a BALB/c mouse to collect peritonealfluid 2 weeks or more thereafter. The antibody produced in theperitoneal fluid was purified using Prosep-G column (manufactured byMillipore) and the like.

As shown in FIG. 10, the thus prepared anti-human BIR1 monoclonalantibody recognized human BIR1 (in the drawing, Clone #170, #68, #95 and#31 represent respective clone of the anti-human BIR1 monoclonalantibody, and 2ndAb represents a negative control, and Anti-FLAG M2represents a positive control).

(3) Preparation of Anti-Human BIR1 Polyclonal Antibody

Solubilized human BIR1L/Fc chimeric protein (100 μg/0.5 ml) was mixedwith the same amount of complete Freund's adjuvant (manufactured byDIFCO) and administered under the dorsal skin of a rabbit. Two weeksthereafter, the antigen (100 μg/0.5 ml) was mixed with the same amountof incomplete Freund's adjuvant (manufactured by DIFCO) and administeredunder the dorsal skin of the rabbit. Two weeks thereafter, test bloodwas collected from the caudal vein, and increase in the antibody titerwas verified by flow cytometry using CHO cells which express the humanBIR1L. When the antibody titer was low, antiserum was prepared byfurther carrying out booster once or twice.

Example 13 Screening of a Compound Which Changes Signal Transduction ofHuman BIR1

In the usual way, 10 μg/mL of rabbit anti-mouse IgG (H+L) antibody(manufactured by Zymed) and 10 μg/ml of the anti-human BIR1 monoclonalantibody prepared in Example 3 were immobilized on a 96 well plate. Atested compound (a low molecular compound, a peptide, an antibody or thelike) was added thereto, and A20IIA1.6 _(ce)ll which stably expressesthe human BIR-1 was inoculated at a density of 1×10⁵ cells/100 μL/well.After culturing at 37° C. for 24 hours, the amount of IL-2 in eachculture supernatant was measured using Quantikine Immunoassay Mouse IL-2ELISA Kit (manufactured by R & D. Systems). A compound which reduces theamount of IL-2, or a compound which increases the same, in comparisonwith a negative control in which the tested compound was not added, isselected as the compound which changes signal transduction of humanBIR1.

Example 14 Screening of a Compound Which Controls Binding of Phosphatase

The phosphorylated peptides (Biotin-HSQELQ³¹³(pY)ATPVF (SEQ ID NO:26) orBiotin-YKSGYV³³⁸(pY)SELNF (SEQ ID NO:28)) derived from 2 ITIM regions ofhuman BIR1 were bound to a 96 well plate immobilized with an anti-biotinantibody (manufactured by Sigma). A tested compound (a low molecularcompound, a peptide, an antibody or the like) was added thereto, andthen a lysate of the A20IIA1.6 cell was added thereto, followed byincubation at 4° C. for 2 hours. After washing with PBS 5 times, primaryantibodies for SHP-1, SHP-2 and SHIP-1 (manufactured by Santacruz) wereadded thereto. After allowing to react at room temperature for 2 hoursand subsequent washing with PBS 5 times, an HRP-labeled secondaryantibody (manufactured by Santacruz) was added thereto. After furthercarrying out the reaction at room temperature for 2 hours, the amount ofphosphatase bound to the phosphorylated peptide was measured using acolor developing kit for peroxidase (manufactured by Sumitomo BakeliteCo., Ltd.). A compound which reduces the amount of phosphatase, or acompound which increases the same, in comparison with a negative controlin which the compound to be tested was not added, was selected.

Example 15 Screening of a Compound Which Changes Expressed Amount ofHuman BIR1

A tested compound (a low molecular compound, a peptide, an antibody orthe like) was added to human monocyte strain THP-1 cells (1×10⁶ cells/2mL) in the presence or absence of LPS (1 μg/mL) and/or IFN-γ (100 ng/mL)and cultured at 37° C. for 24 hours. By extracting total RNA fromrespective cell, the RNA of BIR1 was determined by ABI PRISM 7000Sequence Detection System (manufactured. by Applied Biosystems) usinghuman BIR1-specific primers; 5′-CACAGCCATGGAAGTTGGAATC-3′ (SEQ ID NO:15)and 5′-GAGTGTTTGGCCTCATCTTGG-3′ (SEQ ID NO:16) and QuantiTect SYBR GreenRT-PCR Kit (manufactured by QIAGEN). A compound which reduces the amountof RNA or a compound which increases the same in the presence of LPSand/or IFN-γ, in comparison with a negative control in which the testedcompound was not added, was selected. Alternatively, a compound whichreduces the amount of RNA or a compound which increases the same in theabsence of LPS and IFN-γ, in comparison with a negative control in whichthe tested compound was not added, was selected.

INDUSTRIAL APPLICABILITY

The present invention is markedly useful in developing medicines. Thatis, an antagonist of the protein related to the present invention isuseful in preventing and/or treating a cancer, an immunodeficiencydisease or an infectious disease. Also, an antagonist of the proteinrelated to the present invention is useful in preventing and/or treatingan autoimmune disease, a rejection reaction after organ transplantation,an allergic disease or an inflammatory disease. The screening method ofthe present invention is useful in selecting an antagonist of theprotein related to the present invention.

The protein of the present invention or a partial peptide thereof and anantibody for the protein of the present invention or a partial peptidethereof are useful in preventing and/or treating a cancer, animmunodeficiency disease or an infectious disease as an antagonist ofthe protein related to the present invention. Additionally, the proteinof the present invention or a partial peptide thereof can be used as anantigen in producing the antibody for the protein of the presentinvention or a partial peptide thereof. The polynucleotide of thepresent invention can be used in producing the protein of the presentinvention or a partial peptide thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows expression level of the mRNA of BIR1 in human normaltissues, blood cells and cell lines.

FIG. 2 shows changes in the expression of human BIR1 by variousinflammation stimuli.

FIG. 3 shows a result of the analysis of changes in the expression ofhuman BIR1 in autoimmune disease patient-derived monocytes and B cells(ND: normal donor, vWD: von Willebrand disease, SLE: systemic lupuserythematosus, RA: articular rheumatism, MS: multiple sclerosis, LA: adisease having high value of lupus anticoagulation factor, TA: Takayasuarteritis, ITP: idiopathic thrombocytopenic purpura). *; p<0.05, **;p<0.01, ***; p<0.005.

FIG. 4 shows expression level of FcR chimeric protein in cells whichstably expresses FcR-hBIR1 (wt), FcR-hBIR1 (YWF) and FcR-hKIR2DL3.

FIG. 5 shows inhibition of BCR-mediated increase of intracellular Ca²⁺concentration by human BIR1.

FIG. 6 shows phosphorylation of intracellular tyrosine residue of humanBIR1 and recruited phosphatase, in inhibitory signal transduction (U:Unstimulated, F: F(ab′)₂, I: Intact).

FIG. 7 shows inhibition of BCR-mediated phosphorylation of Erk2 by humanBIR1.

FIG. 8 shows inhibition of BCR-mediated production of IL-2 by humanBIR1.

FIG. 9 shows identification of ITIM domain of human BIR1 and bindingphosphatase.

FIG. 10 shows antigen binding reaction of human BIR1 monoclonalantibody.

1. A method for treating of allergy disease, wherein a pharmaceuticallyeffective amount of the antibody that recognizes both of a proteinconsisting of an amino acid sequence selected from the group consistingof the amino acid sequences represented by SEQ ID NO: 1 to 6 and FcεRIon the cell that expresses said protein is administered to a subject. 2.The method of claim 1, wherein the allergy disease is selected from thegroup consisting of bronchial asthma, atopic dermatitis, nettle rash,allergic rhinitis, allergic conjunctivitis, allergic enterogastritis,anaphylactic shock and food allergy.
 3. The method of claim 1, whereinthe allergy disease is anaphylactic shock.
 4. The method of claim 1,wherein the protein consists of an amino acid sequence selected from thegroup consisting of the amino acid sequences represented by SEQ ID NO: 1to
 4. 5. The method of claim 1, wherein the protein consists of theamino acid sequence represented by SEQ ID NO: 1 or 2.